Ceramide-like lipid-based delivery vehicles and uses thereof

ABSTRACT

Provided herein, in some aspects, are delivery vehicles comprising a ceramide and an agent to be delivered attached to the ceramide. In some embodiments, the ceramide does not comprise a fatty acid (i.e., is a sphingosine). In some embodiments, the ceramide comprises a fatty acid. In some embodiments, the ceramide is a glycoceramide. In some embodiments, the agent is attached to the ceramide covalently (e.g., via a linker). In some embodiments, the agent to be delivered is a therapeutic agent. The ceramide is able to deliver the agent to a cell or to a cellular compartment, as well as across the musical barrier. In some embodiments, agents delivered using the ceramide described herein exhibit longer half-life, compared to agents delivered alone. Methods of delivering a therapeutic agent to a subject for treating a disease using the ceramide delivery vehicle are also provided.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/656,474, filed Apr. 12, 2018, andentitled “CERAMIDE-LIKE. LIPID-BASED DELIVERY VEHICLES AND USESTHEREOF,” the entire contents of which are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with government support under grants Nos. R37DK048106, RO1 DK104868, R21 DK090603, and P30 DK034854, awarded by theNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND

One of the major challenges for applying protein and peptide biologicsto clinical medicine is the lack of rational and efficient methods tocircumvent epithelial and endothelial cell barriers separating largemolecules from target tissues. In the case of epithelial cells liningmucosal surfaces, the pathway for absorption of large solutes (e.g.,biologics) is by transcytosis—a process of transcellular endosometrafficking that connects one surface of the cell with the other.

SUMMARY

The present disclosure, in some aspects, relates to using ceramides(e.g., naturally occurring ceramides and ceramide analogs orceramide-like molecules) as delivery vehicles to deliver agents (e.g.,therapeutic agents) into cells or across epithelial and/or endothelialbarriers. The sorting of the agents via different endocytic pathwaysrelate to the structure of the ceramide. In some embodiments, theceramides are used to deliver agents to a targeted site, e.g., to treata disease in a subject.

Some aspects of the present disclosure provide delivery vehiclescomprising a ceramide and an agent to be delivered, wherein theceramide: (a) does not contain a fatty acid; or (b) comprises a fattyacid of C1-C28; and wherein the agent is attached to the ceramide.

In some embodiments, the ceramide is a ceramide analog. In someembodiments, the ceramide analog is selected from the group consistingof: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide,phytosphingosine, dihydroceramide phosphate, o-acyl-ceramide, ceramidephosphate, sphinganine, and methyl-sphingosine. In some embodiments, theceramide analog comprises an unsaturated hydrocarbon chain attached toornithine, tyrosine, glycine, leucine, proline, glutamine, or taurine.

In some embodiments, the ceramide is a glycoceramide. In someembodiments, the glycoceramide comprises a sugar selected from the groupconsisting of: glucose, galactose, fructose, and GalNac. In someembodiments, the agent to be delivered is attached to the sugar.

In some embodiments, no sugar is attached to the ceramide. In someembodiments, the ceramide is a sphingosine. In some embodiments, theagent to be delivered is attached to the primary hydroxyl group of theceramide. In some embodiments, the agent to be delivered is attached tothe secondary hydroxyl group of the ceramide.

In some embodiments, the agent to be delivered is attached to theceramide via a linker. In some embodiments, the linker is apseudo-glycopeptide linker. In some embodiments, the pseudo-glycopeptidelinker comprises at least one sugar attached to an amino acid backbone.In some embodiments, the at least one sugar is selected from the groupconsisting of: glucose, galactose, and N-Acetylgalactosamine. In someembodiments, the at least one sugar is attached to the amino acidbackbone via a serine side chain.

In some embodiments, the linker is a cleavable linker. In someembodiments, the cleavable linker comprises an ester linkage. In someembodiments, the cleavable linker is a peptide linker comprising anester linkage. In some embodiments, the cleavable linker comprises acleavage motif for an endosomal protease. In some embodiments, theendosomal protease is furin or matriptase. In some embodiments, thelinker is a disulfide linkage.

In some embodiments, the ceramide comprises a fatty acid of C1-C6. Insome embodiments, the ceramide comprises a fatty acid of C4. In someembodiments, the ceramide comprises a fatty acid of C6. In someembodiments, the fatty acid has no double bonds between two carbonatoms. In some embodiments, the ceramide comprises a fatty acid ofC7-C28. In some embodiments, the ceramide comprises a fatty acid of C8.In some embodiments, the fatty acid has at least one cis double bondsbetween two carbon atoms. In some embodiments, the at least one cisdouble bond is in C1-C18 region. In some embodiments, the fatty acidcomprises a chemical moiety in C1-C18 region. In some embodiments, theceramide does not comprise a fatty acid.

In some embodiments, the ceramide comprises a fatty acid of C1-C12.

In some embodiments, the agent to be delivered is selected from thegroup consisting of proteins, peptides, nucleic acids, polysaccharidesand carbohydrates, lipids, glycoproteins, small molecules, syntheticorganic and inorganic drugs exerting a biological effect whenadministered to a subject, and combinations thereof. In someembodiments, the agent to be delivered is a therapeutic agent. In someembodiments, the therapeutic agent is an anti-inflammatory agent, avaccine antigen, a small molecule drug, an anti-cancer drug orchemotherapeutic drug, a clotting factor, a hormone, a steroid, acytokine, an antibiotic, an antibody, a ScFv, a nanobody, a vaccineadjuvant, or a drug for the treatment of a cardiovascular disease, aninfectious disease, an autoimmune disease, allergy, a blood disorder, ametabolic disorder, a skin disease, an eye disease, a lysosomal storagedisease or a neurological disease.

In some embodiments, the agent to be delivered is a protein or apeptide. In some embodiments, the protein or peptide is a vaccineantigen. In some embodiments, the protein or peptide is an antibody, aScFv, or a nanobody. In some embodiments, the protein or peptide is anenzyme. In some embodiments, the enzyme is a lysosomal replacementenzyme. In some embodiments, the protein or peptide is a hormone. Insome embodiments, the protein or peptide is a neurotransmitter. In someembodiments, the protein or peptide is GLP-1, or a functional fragmentthereof. In some embodiments, the protein or peptide is Exendin-4, or afunctional fragment thereof. In some embodiments, the therapeutic agentcomprises GLP-1 or a functional fragment thereof, and Exendin-4 or afunctional fragment thereof. In some embodiments, the therapeutic agentcomprises a ligand for a cell receptor. In some embodiments, the cellreceptor is a growth factor receptor, a G-protein coupled receptor, or atoll-like receptor.

In some embodiments, the therapeutic agent is a nucleic acid.

Other aspects of the present disclosure provide ceramide-therapeuticagent complexes comprising a ceramide and an agent to be delivered,wherein the ceramide: (a) does not comprise a fatty acid; or (b)comprises a fatty acid of C1-C28; and wherein the agent is attached tothe ceramide.

In some embodiments, the ceramide is a ceramide analog. In someembodiments, the ceramide analog is selected from the group consistingof: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide,phytosphingosine, dihydroceramide phosphate, o-acyl-ceramide, ceramidephosphate, sphinganine, and methyl-sphingosine. In some embodiments, theceramide analog comprises an unsaturated hydrocarbon chain attached toornithine, tyrosine, glycine, leucine, proline, glutamine, or taurine.

In some embodiments, the ceramide is a glycoceramide. In someembodiments, the glycoceramide comprises a sugar selected from the groupconsisting of: glucose, galactose, fructose, and GaINAc. In someembodiments, the agent to be delivered is attached to the sugar. In someembodiments, no sugar is attached to the ceramide. In some embodiments,the ceramide is a sphingosine. In some embodiments, the agent to bedelivered is attached to the primary hydroxyl group of the ceramide. Insome embodiments, the agent to be delivered is attached to the secondaryhydroxyl group of the ceramide.

In some embodiments, the agent to be delivered is attached to theceramide via a linker. In some embodiments, the linker is apseudo-glycopeptide linker. In some embodiments, the pseudo-glycopeptidelinker comprises at least one sugar attached to an amino acid backbone.In some embodiments, the at least one sugar is selected In someembodiments, the at least one sugar is attached to the amino acidbackbone via a serine side chain.

In some embodiments, the linker is a cleavable linker. In someembodiments, the cleavable linker comprises an ester linkage. In someembodiments, the cleavable linker is a peptide linker comprising anester linkage. In some embodiments, the cleavable linker comprises acleavage motif for an endosomal protease. In some embodiments, theendosomal protease is furin or matriptase. In some embodiments, thelinker is a disulfide linkage.

In some embodiments, the ceramide comprises a fatty acid of C1-C6. Insome embodiments, the ceramide comprises a fatty acid of C4. In someembodiments, the ceramide comprises a fatty acid of C6. In someembodiments, the fatty acid has no double bonds between two carbonatoms. In some embodiments, the ceramide comprises a fatty acid ofC7-C28. In some embodiments, the ceramide comprises a fatty acid of C8.In some embodiments, the fatty acid has at least one cis double bondsbetween two carbon atoms. In some embodiments, the at least one cisdouble bond is in C1-C18 region. In some embodiments, the fatty acidcomprises a chemical moiety in C1-C18 region. In some embodiments, theceramide does not comprise a fatty acid.

In some embodiments, the ceramide comprises a fatty acid of C1-C12.

In some embodiments, the therapeutic agent is selected from the groupconsisting of proteins, peptides, nucleic acids, polysaccharides andcarbohydrates, lipids, glycoproteins, small molecules, synthetic organicand inorganic drugs exerting a biological effect when administered to asubject, and combinations thereof. In some embodiments, the therapeuticagent is an anti-inflammatory agent, a vaccine antigen, a small moleculedrug, an anti-cancer drug or chemotherapeutic drug, a clotting factor, ahormone, a steroid, a cytokine, an antibiotic, an antibody, a ScFv, ananobody, a vaccine adjuvant, or a drug for the treatment ofcardiovascular disease, an infectious disease, an autoimmune disease,allergy, a blood disorder, a metabolic disorder, a skin disease, an eyedisease, a lysosomal storage disease, or a neurological disease. In someembodiments, the therapeutic agent is a protein or a peptide. In someembodiments, the protein or peptide is a vaccine antigen. In someembodiments, the protein or peptide is an antibody, a ScFv, or ananobody. In some embodiments, the protein or peptide is an enzyme. Insome embodiments, the enzyme is a lysosomal storage enzyme. In someembodiments, the protein or peptide is a hormone. In some embodiments,the protein or peptide is a neurotransmitter. In some embodiments, theprotein or peptide is GLP-1, or a functional fragment thereof. In someembodiments, the protein or peptide is Exendin-4, or a functionalfragment thereof. In some embodiments, the therapeutic agent comprisesGLP-1 or a functional fragment thereof, and Exendin-4 or a functionalfragment thereof. In some embodiments, the therapeutic agent comprises aligand for a cell receptor. In some embodiments, the cell receptor is agrowth factor receptor, a G-protein coupled receptor, or a toll-likereceptor.

In some embodiments, the therapeutic agent is a nucleic acid.

Further provided herein are compositions comprising the deliveryvehicle, or the ceramide-therapeutic agent complex described herein. Insome embodiments, the composition further comprises a pharmaceuticallyacceptable carrier.

Other aspects of the present disclosure provide methods of delivering anagent into a cell, across a mucosal surface, or across an endothelialbarrier the method comprising contacting the delivery vehicle theceramide-therapeutic complex with the cell, the mucosal surface, or theendothelial lumenal surface, under conditions appropriate for uptake ofthe delivery vehicle or the agent into the cell or absorption of thedelivery vehicle or the agent across the mucosal surface or endothelialbarrier.

Other aspects of the present disclosure provide methods of delivering anagent into a cell or across a mucosal or endothelial surface, the methodcomprising contacting the composition described herein with the cell,the mucosal surface, or the endothelial lumenal surface, underconditions appropriate for uptake of the composition or the agent intothe cell or absorption of the composition or the agent across themucosal surface or the endothelial barrier.

Other aspects of the present disclosure provide methods of delivering anagent into a cells, across a mucosal surface, or across an endothelialbarrier in a subject, the method comprising administering to the subjectthe delivery vehicle, the ceramide-therapeutic agent, or the compositiondescribed herein.

Other aspects of the present disclosure provide methods of enhancing thehalf-life of an agent in a subject, the method comprising administeringto the subject the delivery vehicle, the ceramide-therapeutic agent, orthe composition described herein.

Other aspects of the present disclosure provide methods of treating adisease or condition in a subject in need thereof, the method comprisingadministering to the subject an effective amount of the deliveryvehicle, the ceramide-therapeutic agent, or the composition describedherein, wherein the effective amount is an amount sufficient toameliorate/reduce the extent to which the disease or condition occurs inthe subject. In some embodiments, the delivery vehicle, theceramide-therapeutic agent complex, or the composition is administeredparenterally. In some embodiments, the delivery vehicle, theceramide-therapeutic agent complex, or the composition is administeredintravenously, intramuscularly, intradermally, subcutaneously,intrathecally, intraperitoneally, intraarterially, intracardiacally,intraosseously, intraocularly, intravitreally, intranasally orintrapleurally. In some embodiments, the delivery vehicle, theceramide-therapeutic agent complex, or the composition is administerednonparenterally. In some embodiments, the delivery vehicle, theceramide-therapeutic agent complex, or the composition is administeredorally, sublingually, topically, rectally, or via inhalation. Fordelivery across tight endothelial barriers, in some embodiments, theceramide-therapeutic agent complex is delivered intravenously,intramuscularly, or subcutaneously.

The summary above is meant to illustrate, in a non-limiting manner, someof the embodiments, advantages, features, and uses of the technologydisclosed herein. Other embodiments, advantages, features, and uses ofthe technology disclosed herein will be apparent from the DetailedDescription, the Drawings, the Examples and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 . Schematic for trafficking pathway for delivery of peptide orprotein drugs across tight epithelial and endothelial barriers.

FIG. 2 . Schematic of reporter peptide (amino acid sequence: GSGYGRGSGK,SEQ ID NO: 1) fused to the glycoceramide GM1.

FIG. 3 . Structure function studies on GM1 ceramide domain. Non-nativeGM1 species with amplified transcytosis across epithelial barriers invitro were identified. Time course studies and dose dependency of fusionmolecules compared to peptide alone are shown.

FIGS. 4A-4D. In vivo mouse studies. Absorption from intestine into bloodfor GM-fusion molecules 15 and 30 minutes after gastric gavage (FIG.4A), and absorption from intestine into liver for GM-fusion molecules 1hour after gastric gavage (FIG. 4B) are shown. Peptide alone was notabsorbed. Time course for absorption into blood was plotted (FIG. 4C).N=3 independent studies. Schematic of the GM-fusion molecule is shown inFIG. 4D.

FIGS. 5A-5B. Transcytosis of the incretin hormone GLP-1 when fused tothe GM1 transport vehicles across model epithelial barriers in vitro.GLP-1 incretin function is retained after fusion to the oligosaccharidedomain of GM1 (FIG. 5B) (half-log loss in activity). When tested invitro, GLP-1 was transported across epithelial barriers by theshort-chain GM1-species nearly 100-fold above that observed for thepeptide alone (FIG. 5A).

FIG. 6 . In vivo intestinal absorption of GLP-1-GM1 fusion moleculesdelivered by gastric gavage (oral) to mice corrected serum glucoselevels after glucose challenge (glucose tolerance test) at about thesame dose administered by intraperitoneal injection (IP).N=6 independentexperiments.

FIG. 7 . Chemical synthesis of peptide onto ceramide andglucosylceramide. The primary hydroxyl (circle) located on the ceramide(or on sugar of Glc-Cer) are oxidized to form an aldehyde catalyzed bycopper bromide, 2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl (TEMPO) and 2,2bipyridine. The reaction is enhanced in the presence of the basepotassium tert-butoxide (KOtBu). This allows for the reaction toaminooxy containing peptides to form stable bonds (box).

FIG. 8 . GM1 oligosaccharide domain structure and replacement withapproximated peptide-sugar linkers. The structure of the GM1 headgroup(Left) with the sialic acid site for coupling to cargo molecules (blueline). Linker peptides are synthesized via precursor building blocks ofserine attached to different sugar types. Three different linkers aredesigned to test the minimal structure required.

FIG. 9 . Optimization of a furin cleavage motif for incorporation intothe GM1-cargo linker peptide. A fluorescence energy transfer (FRET)based assay was designed by appending paired fluorophores at each end ofthe peptide to be tested. Cleavage of the peptide by recombinantpurified furin was measured as increase in fluorescence (decrease inFRET quenching) in vitro, using buffer alone as negative control (nocleavage) and high dose trypsin as positive control (the furin motif canbe cleaved by other serine proteases). The motif termed FRET 7 was mostefficient.

FIG. 10 . Linkage to ceramide without sugars led to transcytosis acrossepithelial barriers in vitro. Linkage to fatty acid alone (dodecyl-C12)did not. Peptide alone did not cross the epithelial barrier, asexpected.

FIG. 11 . Oxidation of C2-Ceramide. A method called “Parikh-Doering”oxidation was used to oxidize the head group of ceramide to a reactivealdehyde in organic solvent.

FIG. 12 . Reaction of peptide containing aminooxy to the aldehyde group.The “LC9” (D-isomer) linker peptide was coupled using oxime-mediatedreductive amination. LC9-sequence:[alkyne]-[lys-bio]-GSGYGRGSG-[lys-aoa].

FIGS. 13A-13D. HPLC chromatograms of the products generated in FIG. 12 .(FIG. 13A) Control peptide. (FIG. 13B) Crude reaction of ceramide topeptide. (FIG. 13C) After purification. (FIG. 13D) Mass spectrometryanalysis confirmed the presence of peptide linked to ceramide (2+ and 3+charged species).

FIG. 14 . Copper click reaction of Alexa Fluor 488 to ceramide-peptideconjugate.

FIG. 15 . HPLC chromatograms of the ceramide-peptide conjugated preparedin FIG. 14 .

FIG. 16 . Investigating the role of double bond positioning &hydrocarbon chain length of the ceramide fatty acid in lipid packing andsubsequent differential endosomal sorting. GM1 isoforms with identicaloligosaccharide head groups but ceramides of different endogenousstructure, by systematically increasing length and double bond position(C12:0 to C26:1) were synthesized. Partially adopted from Arumugam etal., Essays in Biochemistry 57(1):109-119, incorporated herein byreference.

FIG. 17 Functionalization of GM1 species.

FIG. 18 . Intracellular lipid trafficking—plasma membrane depletiondynamics of GM1 shows transport to the lysosome for some GM1 species.

FIG. 19 . Live cell direct fluorescence images of different GM1 speciesin sorting endosomes and sorting endosome tubules. Table quantifiesdegree of entry into sorting tubules for the different GM1 species.

FIG. 20 . Intracellular lipid sorting-transcytosis and retrogradetrafficking. Shows how the position of the double bond influences entryinto endosome sorting tubules that traffic across the cell bytranscytosis or retrograde into the ER.

FIG. 21 . Confocal fluorescence images of transcytosis in MDCK polarizedcells. Only the short chain GM1 C12:0 species were transcytosed.

FIG. 22 . Knockdown or inhibition of cellular machinery involved intranscytosis by Dyngo-4a chemical inhibition (for Dynamin, left panel),and gene knockdown by siRNA for Exocyst2 for the exocyst complex (rightpanel).

FIG. 23 . Immunostaining images of C6-GM1 peptide or peptide alonetransported across mouse nasal epithelium by 2-photon microscopy (left)and analysis of blood by streptavidin pull-down assay (right).

FIGS. 24A-24H. Ceramide analogs. (FIG. 24A) N-acyloxyacyl-ornitine.(FIG. 24B) Cerilipin. (FIG. 24C) Brominated mololipids. R1, R2=C14 toC20 fatty acid. (FIG. 24D) iso-3-hydroxy heptadecanoic acid-containinglipid. (FIG. 24E) Lipstatin. (FIG. 24F) N-stearoyl proline. (FIG. 24G)Volicitin. (FIG. 24H) N-acyl Taurine.

FIGS. 25A-25C. Confocal fluorescence microscopy images of lysosomaltransport of GM1-peptides. (FIG. 25A) A schematic of endosomal sortingin human microvascular endothelial cells (HMECs). (FIG. 25B) ImagesHMECs incubated either with C16:0-GM1-peptide (top panels), or peptidealone (bottom panel). Cells were treated with 2 μM fluorescently greenlabeled compound for 1 hour for continuous uptake on coverslips, andimaged in the presence of Lysotracker-Red (red). The C16-GM1 lipidcolocalized mostly to lysosomes (yellow puncta), whereas the peptidealone did not enter into cells. (FIG. 25C) C12-GM1 (green) was incubatedfor continuous uptake for 1 hour with HMEC cells and colocalized tolysosomes (yellow), in addition to plasma membrane localization,indicating that this lipid can be sorted both the recycling andlysosomal pathways. The merged image on the left is zoomed in anddisplayed as Merged, green, and red channels on the right. Scale bars=10μm.

FIG. 26 shows the nasal bioavailability of a peptide fused to GM1-6.Apparent bioavailability for peptide fused to GM1-C6:0 was 24.4%compared to the same molecule injected intraperitoneally.Bioavailability of peptide alone was 1.3% compared to the for peptidefused to GM1-C6:0 injected intraperitoneally.

FIG. 27 shows the structure of a panel of ceramides and ceramide analogs(Diene-deoxy-Cer and Dihydro-Cer.

FIG. 28 shows transcytosis of Glc-Cer-C8 and Cer-C6 across MDCKII cells.Efficiency of transcytosis is greater than for peptides fused to GM1 orGM3 species

FIG. 29 shows transcytosis of Glc-Cer-C8 and Cer-C6 across T84intestinal cells. Efficiency of transcytosis is greater than forpeptides fused to GM1 or GM3 species

FIG. 30 shows transcytosis of peptides fused to ceramide-like molecules.They are as efficient as the ceramide-alone vehicles. Fig also showsthat low temperature (4° C.) blocks transport of the indicated ceramidesor ceramide analogs across the MDCKII cell layer, indicating that thetransportation is via transcytosis.

FIG. 31 shows that peptide fused to ceramide-C2, transports across MDCKmonolayer. Peptide fusion to a C12 fatty acid alone, did not transportacross MDCK cells by transcytosis.

FIGS. 32A-32C show that serum half-life is strongly prolonged by fusionto GM1-C4:0. (FIG. 32A) Validation of the fusion molecule in mice. (FIG.38B) Serum levels of intravenously administered GM1-peptide fusion orpeptide alone 1 day after administration. (FIG. 32C) Serum levels ofintravenously administered GM1-peptide fusion or peptide alone 1 dayafter administration.

FIG. 33 shows that several ceramides or ceramide analogs can transportacross MDCK cells and the transport is via transcytosis.

FIG. 34 shows the structures of a panel of glycosphingolipids, ceramidesor ceramide analogs to be conjugated to cargos.

FIG. 35 shows ceramides, linker-ceramides, LC9-linker ceramides, andAF488-LC9-linker ceramides that were tested.

FIG. 36 shows LC9 oximes.

FIG. 37 shows AF488-LC9 oximes.

FIG. 38 shows that ceramide C6 (Cer0C6) is absorbed via the nose in mice15 minutes after dosing.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Delivery of biologically active molecules across tight mucosalepithelial barriers is a major challenge preventing application of mosttherapeutic peptides for oral drug delivery. The sorting endosome sortscargo into four separate pathways: the lysosome pathway, the retrogradepathway to Golgi and ER, the recycling pathway back to the plasmamembrane, and (in polarized cells like epithelial and endothelial cells)into the transcytotic pathway that connects one cell surface with theother, allowing for adsorption. The pathways are distinct and do notintersect with each other (e.g., as described in Saslowsky et al., JBiol Chem. Sep 6; 288(36):25804-9, incorporated herein by reference). Itis possible that lipid sorting into these different pathways may beregulated by the molecular shape of the lipid allowing them to move intohighly-curved membrane buds and tubules that serve the recycling,retrograde and transcytotic pathways. Additionally, there can bespecific sorting into the different pathways via distinct sortingmechanisms.

Glycosphingolipids are present within the outer membrane leaflet of cellmembranes. They contain a ligand-binding oligosaccharide domain thatfaces the extracellular space, and a ceramide domain that anchors thelipid in the membrane bilayer. Ceramides consist of a sphingosine chain(typically C18:1 or C20:1) coupled to a fatty acid that can have diversestructures. The oligosaccharide domain prevents lipid flip-flop betweenmembrane leaflets, causing all the glycosphingolipids to be distributedamong intracellular compartments only by vesicular trafficking. Sortingof proteins and certain sphingolipids to various intracellularcompartments of eukaryotic cells depends on movement of membranesthrough the secretory and endocytic pathways by vesicular carriers. Forproteins, this occurs according to multiple and hierarchically orderedsorting determinants structurally encoded within the protein itself orwithin the structure of an associated receptor or chaperone. Methods ofusing glycosphingolipids isoforms containing a ceramide that comprisesfatty acids of different structures (e.g., different fatty acid chainlength, with or without double bonds) to deliver an agent (e.g., atherapeutic agent) into a cell or across a mucosal harrier have beendescribed (e.g., in U.S. Pat. No. 9,457,097, incorporated herein byreference).

Simplifying the lipid carrier may simplify their synthesis, amplifytheir activity in transport of biologics, and promote their clinicaltranslation. It is known that ceramides alone (e.g., without theoligosaccharide group, or simply glycoceramide) can flip flop from onemembrane leaflet to another (e.g., as described in López-Montero et al.,Biochim Biophys Acta. July; 1798(7):1348-56, incorporated herein byreference). Provided herein, in some aspects, are the use of ceramides(e.g., ceramides alone or glycoceramide) for trafficking agents (e.g.,therapeutic agents) intracellularly or across epithelial or endothelialbarriers.

Accordingly, some aspects of the present disclosure provide deliveryvehicles comprising a ceramide and an agent to be delivered, wherein theceramide: (a) does not comprise a fatty acid; or (b) comprises a fattyacid of C1-C28; and wherein the agent is attached to the ceramide. A“delivery vehicle” refers to a molecule or system that delivers an agent(e.g., a therapeutic agent) to a desired location, e.g., withoutlimitation, to enter a cell or to reach a desired cellular compartment(e.g., the endoplasmic reticulum), to reach a desired part in a subject(e.g., an organ), or to reach a diseased site in a subject (e.g., atumor site). In some embodiments, the delivery vehicle includes theagent to be delivered. In some embodiments, the delivery vehicle isassociated with (or attached to) the agent to be delivered. In thesesituations, complexes comprising the delivery vehicle and the agent tobe delivered are formed and termed herein a “ceramide-agent complex.” Insome embodiments, the agent is a therapeutic agent and the complexcomprising the delivery vehicle and the therapeutic agent is hereintermed a “ceramide-therapeutic agent complex.”

A “ceramide,” as used herein, refers to a molecule comprising asphingosine core structure. A sphingosine is an amino alcohol with anunsaturated hydrocarbon chain that is typically 18-carbon or 20-carbonin length, which forms a primary part of sphingolipids (e.g.,ceramides). The unsaturated hydrocarbon chain is attached to the aminoacid serine to form the sphingosine. The term “ceramide” encompassesnatural ceramides and ceramide analogs (e.g., synthetic or naturalceramide analogs). In some embodiments, the ceramide is a sphingolipidcomposed of sphingosine and a fatty acid. In some embodiments, theceramide of the present disclosure is a ceramide analog. For example,the ceramide of the present disclosure may contain additional chemicalmoieties appended to a natural ceramide or contain modificationscompared to a natural ceramide. Non-limiting examples of ceramideanalogs that may be used in accordance with the present disclosureinclude: 2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide,dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate,sphinganine, and methyl-sphingosine.

In some embodiments, the ceramides of the present disclosure encompassceramide analogs with the core structure built upon amino acids otherthan serine (i.e., having a core structure other than sphingosine). Forexample, in some embodiments, the ceramide described herein comprisesamino acid backbones containing ornithine (e.g., N-acyloxyacyl-ornitine,and bacterial cerilipin, as described in Kawai et al., FEMS Immunol MedMicrobiol 1999, 23, 67 and Tahara et al., Agric Biol Chem 1976, 40, 243,incorporated herein by reference), tyrosine (e.g., Brominated mololipidsfrom sea sponge as described in Ross et al., J Nat Prod 2000, 63, 501,incorporated herein by reference), glycine (e.g., iso-3-hydroxyheptadecanoic acid-containing lipid from Cytophaga johnsonae, asdescribed in Kawazoe et al., J Bacteriol 1991, 173, 5470, incorporatedherein by reference), leucine (e.g., Lipstatin, which is an inhibitor ofpancreatic protease, as described in Weibel et al., J Antibiot 1987,1081, incorporated herein by reference), proline (e.g., N-stearoylproline as described in Sivasamy et al., JAOCS 2001, 78, 897,incorporated herein by reference), glutamine (e.g., Volicitin.N-(17-hydroxylinolenoyl)-1-glutamine, as described in Pare et al., PNAS1998, 95, 13971, incorporated herein by reference) or taurine (e.g.,N-acyl taurine, as described in Saghatelian et al., Biochemistry 2006,45, 9007, incorporated herein by reference). Non-limiting, exemplarystructures of these ceramide analogs are provided in FIGS. 24A-24G.

A “fatty acid” is a carboxylic acid with an aliphatic chain, which iseither saturated or unsaturated. Fatty acids that have double bondsbetween backbone carbons are known as unsaturated. Fatty acids withoutdouble bonds between backbone carbons are known as saturated. The lengthof a fatty acid chain, is herein referred to using the number ofbackbone carbons atoms in the fatty acid chain. For example, a fattyacid chain with X number of backbone carbons is expressed as CX herein,wherein X is an integer. In some embodiments, X is 0, meaning theceramide does not comprise a fatty acid. A ceramide that does not have afatty acid is a sphingosine (also referred to as a “lyso-ceramide”herein). When a sphingosine is referred to herein, it also means themolecule does not have a sugar moiety. In some embodiments, X is aninteger between 1-30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).In some embodiments, the ceramide of the present disclosure comprises afatty acid of C1-C28 (e.g., C1, C2, C3, C4, C5, C6, C7, C8, C9, C10,C11, C12, C13, C14, C15, C16, C17, C18, C19, C20. C21, C22, C23, C24,C25, C26, C27, or C28) in length. The number of double bonds betweenbackbone carbons in a fatty acid chain with X number of backbone carbonsis expressed as CX:Y, wherein Y is the number of double bonds betweenbackbone carbons and is an integer. For example, a fatty acid chain with20 backbone carbons and 1 double bond is expressed as “C20:1” herein. Insome embodiments. Y is 0, meaning the fatty acid does not contain adouble bond between backbone carbons (i.e., a saturated fatty acid). Theceramide chain of the present disclosure, in some embodiments, containsone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) double bondsbetween backbone carbons. In some embodiments, the double bond is acis-double bond. A “cis-double bond” refers to an isoform of a doublebond formed between two carbon atoms. In addition to the double bond,other chemical groups (e.g., —H, —CH3. —COOH) also form bonds with thecarbon atom involved in the cis-double bond, and “cis” indicates thatthe chemical groups other than —H are on the same side of the carbonchain. One skilled in the art is familiar with these terms.

In some embodiments, the ceramide comprises a fatty acid of C1-C12(e.g., for transcytosis applications). In some embodiments, the ceramidecomprises a fatty acid of C13-C28 (e.g., for non-transcytosisapplications).

In some embodiments, the ceramide comprises a fatty acid of C1-C6 (e.g.,C1. C2, C3, C4, C5, or C6) in length. In some embodiments, the fattyacid of C1-C6 has no double bond between two carbon atoms (e.g., any ofthe backbone carbons). In some embodiments, the ceramide comprises afatty acid chain of C4. In some embodiments, the ceramide comprises afatty acid chain of C6. In some embodiments, the fatty acid of C1-C6comprises at least one cis-double bond (e.g., 1, 2, or more cis doublebond).

In some embodiments, the ceramide comprises a fatty acid of C7-C28(e.g., C7. C8, C9. C10. C11, C12, C13. C14, C15, C16. C17. C18, C19,C20, C21. C22. C23, C24. C25. C26. C27, or C28) in length. In someembodiments, the ceramide comprises a fatty acid chain of C8. In someembodiments, the fatty acid of C7-C28 comprises at least one cis-doublebonds between two carbon atoms (e.g., any of the backbone carbons). Forexample, the fatty acid of C7-C28 may comprise 1-10 cis-double bonds. Insome embodiments, the fatty acid of C7-C28 comprises 1-10, 1-9, 1-8,1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 24, 2-3,3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10,5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-10, or9-10 cis-double bonds. In some embodiments, the fatty acid of C7-C28comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cis-double bonds. Insome embodiments, the fatty acid is C7-C16 and comprises at least onecis-double bonds. In some embodiments, the fatty acid is C17-C28 andcomprises at least one cis-double bonds in C1-C18 region of the fattyacid. For example, the at least one cis-double bonds may be in C1-C18,C1-C17, C1-C16, C1-C15. C1-C14, C1-C13, C1-C12, C1-C11. C1-C10, C1-C9,C1-C8. C1-C7, C1-C6, C1-C5, C1-C4. C1-C3, C1-C2, C2-C18, C2-C17, C2-C16.C2-C15, C2-C14. C2-C13, C2-C12, C2-C11, C2-C10. C2-C9, C2-C8. C2-C7.C2-C6, C2-C5, C2-C4, C2-C3. C3-C18. C3-C17, C3-C16, C3-C15. C3-C14,C3-C13, C3-C12. C3-C11, C3-C10. C3-C9, C3-C8, C3-C7, C3-C6. C3-C5,C3-C4, C4-C18, C4-C17, C4-C16. C4-C15, C4-C14. C4-C13, C4-C12. C4-C11.C4-C10, C4-C9, C4-C8, C4-C7, C4-C6, C4-C5, C5-C18, C5-C17. C5-C16,C5-C15. C5-C14, C5-C13. C5-C12, C5-C11. C5-C10, C5-C9. C5-C8. C5-C7,C5-C6. C6-C18, C6-C17, C6-C16, C6-C15. C6-C14, C6-C13, C6-C12. C6-C11,C6-C10. C6-C9, C6-C8, C6-C7, C7-C18. C7-C17, C7-C16. C7-C15, C7-C14.C7-C13, C7-C12. C7-C11. C7-C10, C7-C9. C7-C8, C8-C18, C8-C17, C8-C16.C8-C15, C8-C14. C8-C13, C8-C12. C8-C11, C8-C10, C8-C9. C9-C18. C9-C17,C9-C16. C9-C15, C9-C14. C9-C13. C9-C12, C9-C11. C9-C10, C10-C18.C10-C17. C10-C16, C10-C15, C10-C14, C10-C13, C10-C12, C10-C11, C11-C18,C11-C17, C11-C16, C11-C15. C11-C14, C11-C13, C11-C12, C12-C18. C12-C17,C12-C16, C12-C15, C12-C14, C12-C13, C13-C18, C13-C17, C13-C16, C13-C15,C13-C14, C14-C18. C14-C17, C14-C16, C14-C15. C15-C18, C15-C17. C15-C16,C16-C18. C16-C17, or C17-C18 region of the fatty acid. In someembodiments, the fatty acid is C17-C28 and the at least one cis-doublebonds are in C1-C16 region of the fatty acid. In some embodiments, thefatty acid is C17-C28 and the at least one cis-double bonds are inC1-C14 region of the fatty acid. In some embodiments, the fatty acid ofC7-C28 has no double bond between two carbon atoms (e.g., any of thebackbone carbons).

In some embodiments, the fatty acid of C7-C28 comprises one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical moieties (e.g.,bulky chemical moieties) appended (e.g., covalently attached) to thefatty acid chain. Non-limiting examples of chemical moieties that may beappended to the C7-C28 fatty acid chain of the ceramide described hereininclude: branched methylation or acylation, bulky non-polar fluorophoressuch as BODIPY, aromatic rings, sterols, prenylation, halogenation(e.g., fluorination), and any compound that deviates from the linearstructure of a fully saturated hydrocarbon chain. In some embodiments,the fatty acid is C7-C16 and comprises at least one chemical moieties(e.g., bulky chemical moieties) appended to the fatty acid chain. Insome embodiments, the fatty acid is C17-C28 and comprises at least onechemical moieties (e.g., bulky chemical moieties) appended to the fattyacid chain in C1-C18 region of the fatty acid. For example, the at leastone chemical moieties (e.g., bulky chemical moieties) may be appended inC1-C18, C1-C17. C1-C16, C1-C15, C1-C14. C1-C13, C1-C12. C1-C11. C1-C10,C1-C9. C1-C8, C1-C7. C1-C6. C1-C5, C1-C4, C1-C3, C1-C2, C2-C18, C2-C17.C2-C16, C2-C15. C2-C14, C2-C13, C2-C12, C2-C11, C2-C10, C2-C9, C2-C8,C2-C7, C2-C6, C2-C5, C2-C4, C2-C3. C3-C18, C3-C17. C3-C16, C3-C15,C3-C14, C3-C13. C3-C12, C3-C11. C3-C10, C3-C9. C3-C8. C3-C7, C3-C6.C3-C5, C3-C4. C4-C18, C4-C17. C4-C16, C4-C15, C4-C14. C4-C13, C4-C12.C4-C11, C4-C10. C4-C9, C4-C8. C4-C7, C4-C6, C4-C5. C5-C18, C5-C17.C5-C16, C5-C15. C5-C14, C5-C13, C5-C12. C5-C11. C5-C10, C5-C9. C5-C8.C5-C7, C5-C6. C6-C18, C6-C17. C6-C16. C6-C15. C6-C14, C6-C13. C6-C12,C6-C11. C6-C10, C6-C9. C6-C8. C6-C7, C7-C18. C7-C17, C7-C16. C7-C15,C7-C14. C7-C13, C7-C12. C7-C11. C7-C10. C7-C9, C7-C8, C8-C18. C8-C17,C8-C16, C8-C15. C8-C14. C8-C13, C8-C12. C8-C11, C8-C10. C8-C9, C9-C18.C9-C17. C9-C16. C9-C15, C9-C14. C9-C13, C9-C12. C9-C11. C9-C10, C10-C18.C0-C17, C10-C16. C10-C15. C10-C14. C10-C13. C10-C12. C10-C11. C11-C18.C11-C17. C11-C16. C11-C15. C11-C14. C11-C13, C11-C12. C12-C18, C12-C17.C12-C16, C12-C15. C12-C14, C12-C13. C13-C18. C13-C17, C13-C16. C13-C15,C13-C14. C14-C18, C14-C17. C14-C16. C14-C15, C15-C18, C15-C17. C15-C16,C16-C18. C16-C17, or C17-C18 region of the fatty acid. In someembodiments, the fatty acid is C17-C28 and the at least one chemicalmoieties (e.g., bulky chemical moieties) are appended in C1-C16 regionof the fatty acid. In some embodiments, the fatty acid is C17-C28 andthe at least one chemical moieties (e.g., bulky chemical moieties) areappended in C1-C14 region of the fatty acid.

In some embodiments, any of the ceramide of the present disclosure(e.g., a ceramide with or without a fatty acid chain) further comprisesa sugar (e.g., a glycoceramide). In some embodiments, the ceramidecomprises a fatty acid chain of C1-C6 and further comprises a sugar. Insome embodiments, the ceramide comprises a fatty acid chain of C7-C28and further comprises a sugar. In some embodiments, the ceramide doesnot comprise a fatty acid chain (i.e., a sphingosine or a lyso-ceramide)and further comprises a sugar. A ceramide that does not contain a fattyacid chain but comprises a sugar is also referred to herein as a“glycosphingosine” or a “lyso-glycoceramide.”

A “glycoceramide” refers to a ceramide comprising a sugar attached toits primary hydroxyl group (FIG. 7 ). In some embodiments, the sugar isa simple sugar. A “simple sugar” is a monosaccharide made up of singlesugar molecules. Non-limiting examples of simple sugars include:glucose, fructose, and galactose. Thus, in some embodiments, theceramide of the present disclosure is a glucose-ceramide (Glc-Cer), afructose ceramide, or a galactose ceramide. In some embodiments, thesugar is an oligosaccharide. In some embodiments, the ceramide is aglycoceramide and the agent to be delivered is attached to the sugar ofthe glycoceramide.

In some embodiments, the ceramide of the present disclosure does notcomprise a sugar. In some embodiments, the agent is attached to theceramide (e.g., to the primary hydroxyl group of the ceramide, see FIG.7 ).

The agent may be attached to the ceramide by any methods known in theart. In some embodiments, the agent is attached non-covalently, e.g.,without limitation, by van der Waals forces, hydrophobic interaction,hydrogen bond interaction, or ionic interactions.

In some embodiments, the agent is attached covalently. For example, insome embodiments, the ceramide (e.g., the primary hydroxyl group of theceramide) or the sugar of the glycoceramide may be functionalized with areactive chemical group. One example of such reactive group is a “clickchemistry handle.” Click chemistry is a chemical approach introduceddescribes chemistry tailored to generate substances quickly and reliablyby joining small units together. See, e.g., Kolb, Finn and SharplessAngewandte Chemie International Edition (2001) 40: 2004-2021: Evans,Australian Journal of Chemistry (2007) 60: 384-395). Exemplary couplingreactions (some of which may be classified as “Click chemistry”)include, but are not limited to, formation of esters, thioesters, amides(e.g., such as peptide coupling) from activated acids or acyl halides;nucleophilic displacement reactions (e.g., such as nucleophilicdisplacement of a halide or ring opening of strained ring systems);azide-alkyne Huisgon cycloaddition; thiol-yne addition; imine formation;and Michael additions (e.g., maleimide addition). Non-limiting examplesof a click chemistry handle include an azide handle, an alkyne handle,or an aziridine handle. Azide is the anion with the formula N3-. It isthe conjugate base of hydrazoic acid (HN3). N3—is a linear anion that isisoclectronic with CO2. NCO—, N2O. NO2+ and NCF. Azide can be describedby several resonance structures, an important one being —N═N+═N—. Analkyne is an unsaturated hydrocarbon containing at least onecarbon-carbon triple bond. The simplest acyclic alkynes with only onetriple bond and no other functional groups form a homologous series withthe general chemical formula CnH2n-2. Alkynes are traditionally known asacetylenes, although the name acetylene also refers specifically toC2H2, known formally as ethyne using IUPAC nomenclature. Like otherhydrocarbons, alkynes are generally hydrophobic but tend to be morereactive. Aziridines are organic compounds containing the aziridinefunctional group, a three-membered heterocycle with one amine group(—NH—) and two methylene bridges (—CH2-). The parent compound isaziridine (or ethylene imine), with molecular formula C2H5N.

Other non-limiting, exemplary reactive groups include: acetals, ketals,hemiacetals, and hemiketals, carboxylic acids, strong non-oxidizingacids, strong oxidizing acids, weak acids, acrylates and acrylic acids,acyl halides, sulfonyl halides, chloroformates, alcohols and polyols,aldehydes, alkynes with or without acetylenic hydrogen amides andimides, amines, aromatic, amines, phosphines, pyridines, anhydrides,aryl halides, azo, diazo, azido, hydrazine, and azide compounds, strongbases, weak bases, carbamates, carbonate salts, chlorosilanes,conjugated dienes, cyanides, inorganic, diazonium salts, epoxides,esters, sulfate esters, phosphate esters, thiophosphate esters borateesters, ethers, soluble fluoride salts, fluorinated organic compounds,halogenated organic compounds, halogenating agents, aliphatic saturatedhydrocarbons, aliphatic unsaturated hydrocarbons, hydrocarbons,aromatic, insufficient information for classification, isocyanates andisothiocyanates, ketones, metal hydrides, metal alkyls, metal aryls, andsilanes, alkali metals, nitrate and nitrite compounds, inorganic,nitrides, phosphides, carbides, and silicides, nitriles, nitro, nitroso,nitrate, nitrite compounds, organic, non-redox-active inorganiccompounds, organometallics, oximes, peroxides, organic, phenolic salts,phenols and cresols, polymerizable compounds, quaternary ammonium andphosphonium salts, strong reducing agents, weak reducing agents, acidicsalts, basic salts, siloxanes, inorganic sulfides, organic sulfides,sulfite and thiosulfate salts, sulfonates, phosphonates, organicthiophosphonates, thiocarbamate esters and salts, and dithiocarbamateesters and salts. The agent to be attached to the ceramide (e.g., viathe reactive chemical group) may contain a corresponding chemical groupthat reacts with the oligosaccharide or ceramide, thus resulting incovalent attachment. For example, an agent that is a protein orpolypeptide can be coupled via its N- or C-terminus, or via anendogenous residue (e.g., lysine) by chemical cross-linking.

In some embodiments, the agent is attached to the ceramide via a linker.In some embodiments, the linker is a pseudo-glycopeptide linker (e.g.,see FIG. 8 ). A “pseudo-glycopeptide linker” refers to linkerscomprising at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)sugar (carbohydrate) covalently attached an amino acid backbone (thepeptide) via side chains of the amino acids in the backbone.Non-limiting examples of sugars that may be attached to the amino acidbackbone include: fructose, glucose, galactose, sialic acid, andN-Acetylgalactosamine (GalNAc). In some embodiments, the sugar in thepseudo-glycopeptide linker is attached to the amino acid backbone viathe side chain of a serine, threonine, or asparagine. Forpseudo-glycopeptides that contain more than one sugars, the sugars maybe attached to the amino acid backbone via the same or different aminoacid side chain. In some embodiments, the amino acid backbone of thepseudo-glycopeptide linker comprises 1-30 amino acids. For example, theamino acid backbone of the pseudo-glycopeptide linker may comprise 1-30,1-25, 1-20, 1-15, 1-1-, 1-5, 5-10, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25,10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 amino acids.In some embodiments, the amino acid backbone of the pseudo-glycopeptidelinker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.In some embodiments, the amino acid backbone of the pseudo-glycopeptidelinker comprises 1-10 amino acids. For example, the amino acid backboneof the pseudo-glycopeptide linker may comprise 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9,3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8,5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-10, or 9-10 aminoacids. In some embodiments, the amino acid backbone of thepseudo-glycopeptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids. In some embodiments, the amino acid backbone of thepseudo-glycopeptide linker comprises more than 10 amino acids (e.g.,11-30 amino acids). For example, the amino acid backbone of thepseudo-glycopeptide linker may comprise 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids.

In some embodiments, the linker is a peptide linker. In someembodiments, the agent is a protein or a peptide. In such instances, oneor more of the amino acids of the protein or peptide may be modified toinclude a chemical entity such as a carbohydrate group, a hydroxylgroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for attaching to the oligosaccharide.

In some embodiments, the linker is a cleavable linker. A “cleavablelinker” refers to a linker that can be cleaved by a chemical agent or anenzyme and thus release the agent from the ceramide carrier. In someembodiments, the cleavable linker comprises an ester linkage (e.g., thelinker can be a peptide linker comprising an ester linker). In someembodiments, the ester linkage can be cleaved by an esterase (e.g.,leukocyte esterase whose level is elevated at sites of inflammation, orcarboxylesterase hCE-2 that is specific to gastrointestinal endosomes).

In some embodiments, the cleavable linker comprises a cleavage motif foran endosomal protease. An “endosomal protease” refers to a protease thatis present in endosomes. It is herein interchangeably referred to as a“lysosomal protease.” Endosomal proteases belong to the aspartic,cysteine, or serine proteinase families of hydrolytic enzymes. Endosomalproteases are expressed ubiquitously, and in a tissue- or celltype-specific manner and are usually detected within all vesicles of theendocytic pathway. Reference and classification of endosomal proteasesis available in the art. For example, lists of known endosomal proteasescan be found in the MEROPS database (merops.sanger.ac.uk). In someembodiments, the endosomal protease is furin or matriptase. Furin is acalcium-dependent serine endoprotease that can efficiently cleaveprecursor proteins at their paired basic amino acid processing sites.Furin cleaves proteins downstream of a basic amino acid target sequence(canonically, Arg-X-(Arg/Lys)-Arg′). Matriptase is a trypsin-likeintegral-membrane serine peptidase and cleaves substrates with Arg orLys at the P1 position and prefers small side-chain amino acids, such asAla and Gly, at the P2 position.

In some embodiments, the cleavable linker comprises a disulfide linkage.A “disulfide linkage” is also referred to as a disulfide bond or S—Sbond, which is a covalent bond derived from two thiol groups. Disulfidebonds can be formed by oxidation of sulfhydryl groups and can be cleavedvia reduction (e.g., via using reductants such astris(2-carboxyethyl)phosphine (TCEP), 2-Mercaptoethanol IP-ME) ordithiothreitol (DTT)).

The ceramides described herein are able to act as delivery vehicles todeliver an agent across cell membrane or across mucosal barrier anddirect intracellular trafficking of the agent. For example, in someembodiments, the ceramide-agent complex are directed by the ceramide toa desired intracellular location, e.g., the endoplasmic reticulum (ER).In some embodiments, the ceramide-agent complex is directed by theceramide away from degradative pathways (e.g., lysosome). As such, insome embodiments, the cellular half-life of the agent is prolonged whenthe agent is part of the ceramide-agent complex, compared to when theagent is delivered into cells alone. In some embodiments, the cellularhalf-life of the agent is prolonged by at least 20% when the agent ispart of the ceramide-agent complex, compared to when the agent isdelivered into cells alone. In some embodiments, the cellular half-lifeof the agent is prolonged by at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least100 fold, at least 500 fold, at least 1000 fold or more, when the agentis part of the ceramide-agent complex, compared to when the agent isdelivered into cells alone. In some embodiments, the cellular half-lifeof the agent is prolonged by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 2 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, 100 fold, 500 fold, 1000 fold or more,when the agent is part of the ceramide-agent complex, compared to whenthe agent is delivered into cells alone.

In some embodiments, the agent is delivered across an epithelial orendothelial barrier (e.g., a mucosal barrier) by transcytosis, when theagent is attached to the ceramide to form a ceramide-agent complex.Mucosal barrier is composed of compact epithelial cell lining (e.g., inthe stomach or in the intestines). The intestinal mucosal harrier, alsoreferred to as intestinal barrier, refers to the property of theintestinal mucosa that ensures adequate containment of undesirableluminal contents within the intestine while preserving the ability toabsorb nutrients. The gastric mucosal barrier is the property of thestomach that allows it to safely contain the gastric acid required fordigestion.

In some embodiments, the agent is not able to cross an epithelial orendothelial barrier (e.g., a mucosal barrier) alone and is able to crossmucosal barriers in complex with the ceramides described herein. In someembodiments, the delivery of the agent across an epithelial orendothelial barrier (e.g., a mucosal barrier) is enhanced (e.g., by atleast 20%) when the agent is in complex with the ceramides describedherein, compared to when the agent is delivered alone. For example, thedelivery of the agent across an epithelial or endothelial harrier (e.g.,a mucosal barrier) is enhanced by at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold,at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold,at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold,at least 100 fold, at least 500 fold, at least 1000 fold or more, whenthe agent is in complex with the ceramides described herein, compared towhen the agent is delivered alone. In some embodiments, the delivery ofthe agent across an epithelial or endothelial barrier (e.g., a mucosalbarrier) is enhanced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2fold, 5 fold, 10 fold, 20 fold, 30 fold. 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, 100 fold, 500 fold, 1000 fold or more, when theagent is in complex with the ceramides described herein, compared towhen the agent is delivered alone.

Other aspects of the present disclosure provide agents that may bedelivered by the ceramides described herein. The agent may be anybioactive agent or therapeutic agent. A “therapeutic agent” refers to anagent that has therapeutic effects to a disease or disorder. The complexbetween the ceramide and the therapeutic agent is referred to herein asthe “ceramide-therapeutic agent complex.” A therapeutic agent may be,without limitation, proteins, peptides, nucleic acids, small moleculesdrugs, polysaccharides and carbohydrates, lipids, glycoproteins, smallmolecules, synthetic organic and inorganic drugs exerting a biologicaleffect when administered to a subject, and combinations thereof. In someembodiments, the therapeutic agent is an anti-inflammatory agent, avaccine antigen, a vaccine adjuvant, an antibody, a ScFv, a nanobody,and enzyme, an anti-cancer drug or chemotherapeutic drug, a clottingfactor, a hormone, a steroid, a cytokine, an antibiotic, or a drug forthe treatment of cardiovascular disease, an infectious disease, anautoimmune disease, allergy, a blood disorder, a metabolic disorder, askin disease, an eye disease, a lysosomal storage disease, or aneurological disease. In some embodiments, the therapeutic agent is aprotein or a peptide. In some embodiments, the protein or peptide isglucagon-like peptide-1 (GLP-1), or a functional fragment thereof. Insome embodiments, the protein or peptide is Exendin-4, or a functionalfragment thereof.

“Glucagon-like peptide-1 (GLP-1)” is a 30 amino acid long peptidehormone deriving from the tissue-specific posttranslational processingof the proglucagon gene. It is produced and secreted by intestinalenteroendocrine L-cells and certain neurons within the nucleus of thesolitary tract in the brainstem upon food consumption. The initialproduct GLP-1(1-37) is susceptible to amidation and proteolytic cleavagewhich gives rise to the two truncated and equipotent biologically activeforms, GLP-1(7-36)amide and GLP-1(7-37). Active GLP-1 composes twoα-helices from amino acid position 13-20 and 24-35 separated by a linkerregion. GLP-1 possesses several physiological properties that make it(and its functional analogs) a subject of intensive investigation as apotential treatment of diabetes mellitus. Further, GLP-1 is has theability to decrease blood sugar levels in a glucose-dependent manner byenhancing the secretion of insulin. Thus, GLP-1 has been associated withnumerous regulatory and protective effects. GLP-1-based treatment hasbeen associated with weight loss and lower hypoglycemia risks, two veryimportant aspects of a life with diabetes.

“Exendin-4” is a peptide agonist of the glucagon-like peptide (GLP)receptor that promotes insulin secretion. Exendin-4 binds to the intacthuman Glucagon-like peptide-1 receptor (GLP-1R) in a similar way toGLP-1 and bears a 50% amino acid homology to GLP-1. Exendin-4facilitates glucose control via augmentation of pancreas response (i.e.increases insulin secretion) in response to eating meals, suppressingpancreatic release of glucagon in response to eating, reducing rate ofgastric emptying, suppressing appetite, and reducing liver fat content.In some embodiments, the therapeutic agent is a fusion protein compriseGLP-1 or a functional fragment thereof and Exendin-4 or a functionalfragment thereof.

In some embodiments, the therapeutic agent is a vaccine antigen. A“vaccine antigen” is a molecule or moiety that, when administered to asubject, activates or increases the production of antibodies thatspecifically bind the antigen. In some embodiments, an antigen is aprotein or a polysaccharide. Antigens of pathogens are well known tothose of skill in the art and include, but are not limited to parts(coats, capsules, cell walls, flagella, fimbriae, and toxins) ofbacteria, viruses, and other microorganisms. A vaccine typicallycomprises an antigen, and is intentionally administered to a subject toinduce an immune response in the recipient subject. The antigen may befrom a pathogenic virus, bacteria, or fungi.

Examples of pathogenic virus include, without limitation: Retroviridae(e.g., human immunodeficiency viruses, such as HIV-1 (also referred toas HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, suchas HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Caliciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruscs, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses'); Poxviridae (variola viruses, vaccinia viruses, pox viruses);and Iridoviridae (e.g., African swine fever virus); and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1=internally transmitted; class 2=parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of pathogenic bacteria include, without limitation:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare,M. kansasii, M. gordonae). Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus). Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group). Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diphtheriae,Corynebacterium sp.,

Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida. Bacteroides sp., Fusobacterium nucleatum. Streptobacillusmoniliformis, Treponema pallidum, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of pathogenic fungi include, without limitation: Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis. Chlamydia trachomatis, Candida albicans. Other infectiousorganisms (i.e., protists) include: Plasmodium falciparum and Toxoplasmagondii.

In some embodiments, the therapeutic agent is an agent that inducesimmunological tolerance. Immunologic tolerance is a state of immuneunresponsiveness specific to a particular antigen or set of antigensinduced by previous exposure to that antigen or set. In someembodiments, the immunologic tolerance is oral tolerance. Oral toleranceis the state of local and systemic immune unresponsiveness that isinduced by oral administration of innocuous antigen such as foodproteins. In some embodiments, the therapeutic agent is an agent forinduce immunological tolerance for the treatment of allergy orautoimmune disease (e.g., multiple sclerosis).

Other non-limiting examples of agents that may be delivered using theceramides described herein are provided.

Non-limiting, exemplary chemopharmaceutically compositions that may beused in the liposome drug delivery systems of the present disclosureinclude. Actinomycin, All-trans retinoic acid, Azacitidine,Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine,Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin,Docetaxel. Doxifluridine, Doxorubicin. Epirubicin, Epothilone,Etoposide. Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib,Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan,Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine.

Examples of antineoplastic compounds include, without limitation:nitrosoureas, e.g., carmustine, lomustine, semustine, strepzotocin;Methylhydrazines, e.g., procarbazine, dacarbazine; steroid hormones,e.g., glucocorticoids, estrogens, progestins, androgens,tetrahydrodesoxycaricosterone, cytokines and growth factors;Asparaginase.

Examples of immunoactive compounds include, without limitation:immunosuppressives, e.g., pyrimethamine, trimethopterin, penicillamine,cyclosporine, azathioprine; immunostimulants, e.g., levamisole, diethyldithiocarbamate, enkephalins, endorphins.

Examples of antimicrobial compounds include, without limitation:antibiotics, e.g., beta lactam, penicillin, cephalosporins, carbapenimsand monobactams, beta-lactamase inhibitors, aminoglycosides, macrolides,tetracyclins, spectinomycin; Antimalarials, Amebicides.

Antiprotazoal. Antifungals, e.g., amphotericin beta or clotrimazole,antiviral. e.g., acyclovir, idoxuridine, ribavirin, trifluridine,vidarbine, gancyclovir.

Examples of parasiticides include, without limitation: antihalmintics,Radiopharmaceutics, gastrointestinal drugs.

Examples of hematologic compounds include, without limitation:immunoglobulins; blood clotting proteins; e.g., antihemophilic factor,factor IX complex; anticoagulants, e.g., dicumarol, heparin Na;fibrolysin inhibitors, tranexamic acid.

Examples of cardiovascular drugs include, without limitation: peripheralantiadrenergic drugs, centrally acting antihypertensive drugs. e.g.,methyldopa, methyldopa HCl; antihypertensive direct vasodilators, e.g.,diazoxide, hydralazine HCl; drugs affecting renin-angiotensin system;peripheral vasodilators, phentolamine; antianginal drugs; cardiacglycosides; inodilators; e.g., amrinone, milrinone, enoximone,fenoximone, imazodan, sulmazole; antidysrhythmic; calcium entryblockers; drugs affecting blood lipids; nadolol, hosentan, rezulin.

Examples of respiratory drugs include, without limitation:sypathomimetic drugs: albuterol, bitolterol mesylate, dobutamine HCl,dopamine HCl, ephedrine SO, epinephrine, fenfluramine HCl, isoproterenolHCl, methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl,ritodrine HCl; cholinomimetic drugs, e.g., acetylcholine Cl;anticholinesterases, e.g., edrophonium Cl; cholinesterase reactivators;adrenergic blocking drugs, e.g., acebutolol HCl, atenolol, esmolol HCl,labetalol HCl, metoprolol, nadolol, phentolamine mesylate, propanololHCl; antimuscarinic drugs. e.g., anisotropine methylbromide, atropineSO4, clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr.

Examples of neuromuscular blocking drugs include, without limitation:depolarizing, e.g., atracurium besylate, hexafluorenium Br, metocurineiodide, succinylcholine Cl, tubocurarine C1, vecuronium Br; centrallyacting muscle relaxants, e.g., baclofen.

Examples of neurotransmitters and neurotransmitter agents include,without limitation: acetylcholine, adenosine, adenosine triphosphate,amino acid neurotransmitters, e.g., excitatory amino acids, GABA,glycine; biogenic amine neurotransmitters, e.g., dopamine, epinephrine,histamine, norepinephrine, octopamine, serotonin, tyramine;neuropeptides, nitric oxide, K+ channel toxins.

Examples of antiparkinson drugs include, without limitation: amaltidineHCl, benztropine mesylate, e.g., carbidopa.

Examples of diuretic drugs include, without limitation:dichlorphenamide, methazolamide, bendroflumethiazide, polythiazide.

Examples of uterine, antimigraine drugs include, without limitation:carboprost tromethamine, mesylate, methysergide maleate.

Examples of hormones include, without limitation: pituitary hormones,e.g., chorionic gonadotropin, cosyntropin, menotropins, somatotropin,iorticotropin, protirelin, thyrotropin, vasopressin, lypressin; adrenalhormones, e.g., beclomethasone dipropionate, betamethasone,dexamethasone, triamcinolone; pancreatic hormones, e.g., glucagon,insulin; parathyroid hormone, e.g., dihydrochysterol; thyroid hormones,e.g., calcitonin etidronate disodium, levothyroxine Na, liothyronine Na,liotrix, thyroglobulin, teriparatide acetate; antithyroid drugs;estrogenic hormones; progestins and antagonists, hormonalcontraceptives, testicular hormones; gastrointestinal hormones;cholecystokinin, enteroglycan, galanin, gastric inhibitory polypeptide,epidermal growth factor-urogastrone, gastric inhibitory polypeptide,gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin,motilin, peptide YY, secretin, vasoactive intestinal peptide, sincalide.

Examples of enzymes include, without limitation: lysosomal storageenzymes, hyaluronidase, streptokinase, tissue plasminogen activator,urokinase, PGE-adenosine deaminase, oxidoreductases, transferases,polymerases, hydrolases, lyases, synthases, isomerases, and ligases,digestive enzymes (e.g., proteases, lipases, carbohydrases, andnucleases). In some embodiments, the enzyme is selected from the groupconsisting of lactase, beta-galactosidase, a pancreatic enzyme, anoil-degrading enzyme, mucinase, cellulase, isomaltase, alginase,digestive lipases (e.g., lingual lipase, pancreatic lipase,phospholipase), amylases, cellulases, lysozyme, proteases (e.g., pepsin,trypsin, chymotrypsin, carboxypeptidase, elastase), esterases (e.g.sterol esterase), disaccharidases (e.g., sucrase, lactase,beta-galactosidasc, maltasc, isomaltasc), DNascs, and RNascs.

Examples of intravenous anesthetics include, without limitation:droperidol, etomidate, fetanyl citrate/droperidol, hexobarbital,ketamine HCl, methohexital Na, thiamylal Na, thiopental Na.

Examples of antiepileptics include, without limitation, carbamazepine,clonazepam, divalproex Na, ethosuximide, mephenytoin, paramethadione,phenytoin, primidone.

Examples of peptides and proteins that may be used as therapeutic agentsinclude, without limitation: ankyrins, arrestins, bacterial membraneproteins, clathrin, connexins, dystrophin, endothelin receptor,spectrin, selectin, cytokines; chemokines; growth factors, insulin,erythropoietin (EPO), tumor necrosis factor (TNF), neuropeptides,neuropeptide Y, neurotensin, transforming growth factor alpha,transforming growth factor beta, interferon (IFN), and hormones, growthinhibitors, e.g., genistein, steroids etc; glycoproteins, e.g., ABCtransporters, platelet glycoproteins. GP1b-IX complex, GP11b-IIIacomplex, vitronectin, thrombomodulin. CD4, CD55, CD58. CD59, CD44,lymphocye function-associated antigen, intercellular adhesion molecule,vascular cell adhesion molecule, Thy-1, antiporters. CA-15-3 antigen,fibronectins, laminin, myelin-associated glycoprotein. GAP, GAP-43,Exendin-4, and GLP-1.

Examples of cytokines and cytokine receptors include, withoutlimitation: interleukin-1 (IL-1). IL-2. IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9. IL-10, IL-11, IL-12, IL-13, IL-14, IL-15. IL-16. IL-17.IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5receptor, IL-6 receptor. IL-7 receptor, IL-8 receptor, IL-9 receptor,IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18receptor, lymphokine inhibitory factor, macrophage colony stimulatingfactor, platelet derived growth factor, stem cell factor, tumor growthfactor beta, tumor necrosis factor, lymphotoxin. Fas, granulocyte colonystimulating factor, granulocyte macrophage colony stimulating factor,interferon-alpha, interferon-beta, interferon-gamma.

Examples of growth factors and protein hormones include, withoutlimitation: erythropoietin, angiogenin, hepatocyte growth factor,fibroblast growth factor, keratinocyte growth factor, nerve growthfactor, tumor growth factor-alpha, thrombopoietin, thyroid stimulatingfactor, thyroid releasing hormone, neurotrophin, epidermal growthfactor. VEGF, ciliary neurotrophic factor, LDL, somatomedin, insulingrowth factor, insulin-like growth factor I and II.

Examples of chemokines include, without limitation: ENA-78, ELC,GRO-alpha, GRO-beta, GRO-gamma, HRG, LIF, IP-10, MCP-1. MCP-2, MCP-3,MCP-4, MIP-lalpha, M1P-1beta. M1G, MDC, NT-3, NT-4. SCF, LIF, lcptin,RANTES, lymphotactin, cotaxin-1, eotaxin-2, TARC. TECK. WAP-1. WAP-2.GCP-1, GCP-2; alpha-chemokine receptors: CXCR1. CXCR2. CXCR3, CXCR4,CXCR5. CXCR6, CXCR7; beta-chemokine receptors: CCR1, CCR2, CCR3, CCR4,CCR5. CCR6, CCR7.

In some embodiments, antibodies that may be delivered using the deliveryvehicle described herein target antigens including, without limitation:(a) anti-cluster of differentiation antigen CD-1 through CD-166 and theligands or counter receptors for these molecules; (b) anti-cytokineantibodies, e.g., anti-IL-1 through anti-IL-18 and the receptors forthese molecules; (c) anti-immune receptor antibodies, antibodies againstT cell receptors, major histocompatibility complexes I and R, B cellreceptors, selectin killer inhibitory receptors, killer activatingreceptors, OX-40, MadCAM-1, Gly-CAM1, integrins, cadherens,sialoadherens, Fas, CTLA-4, Fc.gamma.-receptors, Fcalpha-receptors,Fc.epsilon.-receptors, Fc.mu.-receptors, and their ligands; (d)anti-metalloproteinase antibodies, e.g., collagenase. MMP-1 throughMMP-8, TIMP-1, TIMP-2; anti-cell lysis/proinflammatory molecules, e.g.,perforin, complement components, prostanoids, nitron oxide,thromboxanes; and (e) anti-adhesion molecules. e.g., carcioembryonicantigens, lamins, fibronectins.

Non-limiting, exemplary antibodies and fragments thereof include:bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®,indicated for B cell chronic lymphocytic leukemia), gemtuzumab(MYLOTARG®, hP67.6, anti-CD33, indicated for leukemia such as acutemyeloid leukemia), rituximab (RITUXAN®), tositumomab (BEXXAR®,anti-CD20, indicated for B cell malignancy). MDX-210 (bispecificantibody that binds simultaneously to HER-2/neu oncogene protein productand type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)),oregovomab (OVAREX®, indicated for ovarian canccr), cdrecolomab(PANOREX®), daclizumab (ZENAPAX®), palivizumab (SYNAGIS®, indicated forrespiratory conditions such as RSV infection), ihritumomah tiuxetan(ZEVALIN®, indicated for Non-Hodgkin's lymphoma), cetuximab (ERBITUiX®),MDX-447. MDX-22, MDX-220 (anti-TAG-72). IOR-C5, IOR-T6 (anti-CD1), IOREGF/R3, celogovab (ONCOSCINT® OV103), epratuzumab (LYMPHOCIDE®),pemtumomab (THERAGYN®) and Gliomab-H (indicated for brain cancer,melanoma). Other antibodies and antibody fragments are contemplated andmay be used in accordance with the disclosure. In some embodiments, thetherapeutic agent is a nanobody. A “nanobody” is a therapeutic proteinbased on single-domain antibody fragments that contain the uniquestructural and functional properties of naturally-occurring heavy chainonly antibodies. In some embodiments, the nanobody is anti-inflammatory.In some embodiments, the nanobody is against pathogenic agents (e.g.,anthrax).

In some embodiments, the therapeutic agent is a ligand for a cellreceptor (e.g., without limitation, a growth factor receptor, aG-protein coupled receptor, or a toll-like receptor).

A regulatory protein may be, in some embodiments, a transcription factoror a immunoregulatory protein. Non-limiting, exemplary transcriptionalfactors include: those of the NFkB family, such as Rel-A, c-Rel, Rel-B,p50 and p52; those of the AP-1 family, such as Fos, FosB, Fra-1, Fra-2.Jun, JunB and JunD; ATF; CREB; STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2and -4; MAF; Thyroid Factor, IRF; Oct-1 and -2; NF-Y; Egr-1; and USF-43,EGR1, Sp1, and E2F1.

Examples of antiviral agents include, without limitation: reversetranscriptase inhibitors and nucleoside analogs. e.g. ddI, ddC, 3TC,ddA. AZT; protease inhibitors, e.g., Invirase, ABT-538; inhibitors of inRNA processing, e.g., ribavirin.

Other non-limiting examples of known therapeutics which may be deliveredby coupling to a ceramide described herein include:

-   -   (a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,        Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime. VePesid,        Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS,        Estrace. Glucophage (Bristol-Myers Squibb);    -   (b) Ceclor, Lorabid. Dynabac. Prozac. Darvon, Permax, Zyprexa.        Humalog, Axid, Gemzar. Evista (Eli Lily);    -   (c) Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide,        Plendil, Cozaar/Hyzaar, Pepcid. Prilosec, Primaxin, Noroxin,        Recombivax HB, Varivax, Timoptic/XE. Trusopt, Proscar, Fosamax,        Sinemet, Crixivan, Propecia, Vioxx, Singulair, Maxalt,        Ivermectin (Merck & Co.);    -   (d) Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, Procardia        XL, Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox,        Glucotrol XL, Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept,        Lipitor (Pfizer);    -   (e) Vantin, Rescriptor, Vistide, Genotropin,        Micronase/Glyn./Glyb., Fragmin, Total Medrol, Xanax/alprazolam,        Sermion, Halcion/triazolam, Freedox, Dostinex, Edronax, Mirapex,        Pharmorubicin, Adriamycin, Camptosar, Remisar, Depo-Provera,        Caverject, Detrusitol, Estring, Healon, Xalatan, Rogaine        (Pharmacia & Upjohn);    -   (f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin,        Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT,        Suramin, Clinafloxacin (Warner Lambert).

Non-limiting examples of therapeutic agents for eye diseases include:Anti-infective drugs (e.g., Aciclovir, Chloramphenicol, Ciprofloxacin.Gentamicin, Neomycin. Polymyxin B); Anti-inflammatory drugs (e.g.,Betamethasone. Dexamethasone, Emedastine, Nedocromil sodium,Prednisolone. Sodium cromoglicate); Artificial tears (e.g., Carmellose,Hydroxyethylcellulose. Hypromellose. Polyvinyl alcohol); and Mydriatics(e.g., Atropine, cyclopentolate, Phenylephrine).

Further non-limiting examples of therapeutic agents which may bedelivered by the ceramide-therapeutic agent complex of the presentinvention may be found in: Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 9th ed. McGraw-Hill 1996, incorporated herein byreference.

The delivery vehicle comprising a ceramide and an agent to be delivered,or a ceramide-agent complex (e.g., a ceramide-therapeutic agent) complexmay be formulated into pharmaceutical compositions. In some embodiments,the pharmaceutical composition further comprises a pharmaceuticallyacceptable carrier. “Pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. A “pharmaceuticallyacceptable carrier” may be a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agents from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the tissue of the patient (e.g.,physiologically compatible, sterile, physiologic pH, etc.). The term“carrier” denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being co-mingled with the molecules of the presentdisclosure, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficacy. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. The term “unit dose” when used in reference to apharmaceutical composition of the present disclosure refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The formulation of the pharmaceutical composition may dependent upon theroute of administration. Injectable preparations suitable for parenteraladministration or intratumoral, peritumoral, intralesional orperilesional administration include, for example, sterile injectableaqueous or oleaginous suspensions and may be formulated according to theknown art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution, suspension or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in 1.3propanediol or 1,3 butandiol.

Among the acceptable vehicles and solvents that may be employed arewater, Ringers solution, U.S.P. and isotonic sodium chloride solution.In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed including synthetic mono- or di-glycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables. The injectable formulations can be sterilized, for example,by filtration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

In some embodiments, the pharmaceutical compositions used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Alternatively, preservatives can be used toprevent the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Thepharmaceutical composition ordinarily will be stored in lyophilized formor as an aqueous solution if it is highly stable to thermal andoxidative denaturation. The pH of the preparations typically will beabout from 6 to 8, although higher or lower pH values can also beappropriate in certain instances.

Other aspects of the present disclosure provide methods of delivering anagent (e.g., a therapeutic agent) into a cell or across a mucosalsurface or an endothelial barrier, the method comprising contacting thedelivery vehicle, the ceramide-agent complex (e.g., theceramide-therapeutic agent complex), or the pharmaceutical compositioncomprising the delivery vehicle or the ceramide-agent complex (e.g., theceramide-therapeutic agent complex) with the cell, the mucosal surface,or the endothelial lumenal surface, under conditions appropriate foruptake of the delivery vehicle or the agent into the cell or absorptionof the delivery vehicle or the agent across the mucosal surface or theendothelial barrier (e.g., via transcytosis). In some embodiments, thedelivery vehicle, the ceramide-agent complex, or the pharmaceuticalcomposition comprising the delivery vehicle or the ceramide-agentcomplex (e.g., the ceramide-therapeutic agent complex) are administeredto a subject.

Methods of delivering agents (e.g., therapeutic agents) to differentorgans via intravenous infusion of the delivery vehicle or theceramide-agent complex are also provided. Such organs may be, forexample, without limitation, skeleton, joints, muscles, tendons, varioustypes of glands, oesophagus, stomach, small intestine, large intestine,liver, pancreas, pharynx, larynx, trachea, bronchi, lungs, diaphragm,kidneys, ureters, bladder, urethra, ovaries, uterus, prostate, heart,lymph nodes, bone marrow, thymus, spleen, brain, and spinal cord. Insome embodiments, the delivery vehicle or the ceramide-agent complex isdelivered across an endothelial barrier (e.g., the endothelial barrierin the heart or brain) when it reaches the organ after intravenousinfusion. In some embodiments, the intravenously infused deliveryvehicle or the ceramide-agent complex are delivered to different organsand are sorted into intracellular compartments such as the lysosome(e.g., for lysosomal replacement therapies) or ER (e.g., for addressingprotein folding problems). Delivery to liver after intravenous infusionof the delivery vehicle or the ceramide-agent complex greatly enhances(e.g., by at least 20%) the delivery of the agent to the liver, comparedto delivering the agent alone.

Methods of enhancing the half-life of an agent in a subject areprovided, the method comprising administering to the subject thedelivery vehicle, the ceramide-therapeutic agent complex, or thecomposition described herein.

Methods of treating a disease or condition in a subject in need thereofare provided, the method comprising administering to the subject aneffective amount of the delivery vehicle, the ceramide-therapeutic agentcomplex, or the composition described herein, wherein the effectiveamount is an amount sufficient to ameliorate/reduce the extent to whichthe disease or condition occurs in the subject. The disease may be anydisease that can be treated by the agents described herein. In someembodiments, the disease is infection, allergy, autoimmune diseases(e.g., multiple sclerosis), liver diseases, lung diseases, neurologicaldiseases, eye diseases or cancer.

An “effective amount” is the amount necessary or sufficient to have adesired effect in a subject. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated, the severity of the condition, the durationof the treatment, the nature of the concurrent therapy (if any), thespecific route of administration and other factors within the knowledgeand expertise of the health care practitioner. For example, an effectiveamount could be that amount necessary to eliminate a tumor, cancer, orbacterial, viral or fungal infection. This amount will vary fromindividual to individual and can be determined empirically using knownmethods by one of ordinary skill in the art.

The delivery vehicle, the ceramide-agent complex, or the pharmaceuticalcomposition comprising the delivery vehicle or the ceramide-agentcomplex (e.g., the ceramide-therapeutic agent complex) may beadministered by any route. Routes of administration include enteralroutes, such as oral and any other means by which the gastrointestinaltract is involved, and parenteral routes, such as by injection(subcutaneous, intravenous, intramuscular injection) or infusion(typically by intravenous route). In some embodiments, theadministration is done intravenously, intramuscularly, intradermally,subcutaneously, intrathecally, intraperitoneally, intraarterially,intracardiacally, intraosseously, intraocularly, intravitreally,intrapleurally, intranasally, or injection into the joint. The injectioncan be in a bolus or a continuous infusion. In some embodiments, theadministration is done nonparenterally (e.g., done orally, sublingually,topically, rectally, via inhalation, nasally, as eye drops, as eyepatches, to the cervix, or to the skin). For delivery across tightendothelial barriers, in some embodiments, the ceramide-therapeuticagent complex is delivered intravenously, intramuscularly, orsubcutaneously.

Methods of treating a disease or disorder are also provided. Thedelivery vehicle, the ceramide-agent complex, or the pharmaceuticalcomposition comprising the delivery vehicle or the ceramide-agentcomplex (e.g., the ceramide-therapeutic agent complex) may beadministered to a subject who has, has had or is susceptible todeveloping one or more conditions/diseases that require or would benefitfrom treatment. For example, the compositions described herein may beused to treat, prevent or ameliorate immune system deficiencies,infectious diseases (viral, fungal, bacterial or parasitic), autoimmunediseases, diabetes, blood disorders, cancers, metabolic disorders,allergies, inflammatory bowel disease and skin disorders. In addition,gangliosides attached to antigen can be administered to stimulate asubject's response to a vaccine. The antigen is selected from the groupconsisting of: an antigen that is characteristic of a pathogen, anantigen that is characteristic of an autoimmune disease, an antigen thatis characteristic of an allergen and an antigen that is characteristicof a tumor. In some embodiments, the disease or disorder to be treatedis diabetes. In some embodiments, the disease or disorder is infection,e.g., by a pathogenic virus, bacteria, or fungi. In some embodiments,the disease or disorder is cancer.

Immune system deficiencies include any disease or disorder in which asubject's immune system is not functioning normally or in which it wouldbe useful to boost the subject's immune response, for example toeliminate a tumor or cancer (e.g. tumors of the brain, lung (e.g. smallcell and non-small cell), ovary, breast, prostate, colon, as well asother carcinomas and sarcomas) or an infection in a subject.

Examples of autoimmune diseases include, without limitation: Addison'sdisease, diabetes mellitus (type 1). Graves' disease, interstitialcystitis, lupus erythematous, multiple sclerosis and Hashimoto'sthyroiditis. Allergic conditions include eczema, allergic rhinitis orcoryza, hay fever, bronchial asthma, urticaria (hives) and foodallergies, and other atopic conditions.

Non-limiting, exemplary cancers include: neoplasms, malignant tumors,metastases, or any disease or disorder characterized by uncontrolledcell growth such that it would be considered cancerous. The cancer maybe a primary or metastatic cancer. Cancers include, but are not limitedto, adult and pediatric acute lymphoblastic leukemia, acute myeloidleukemia, adrenocortical carcinoma. AIDS-related cancers, anal cancer,cancer of the appendix, astrocytoma, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma,fibrous histiocytoma, brain cancer, brain stem glioma, cerebellarastrocytoma, malignant glioma, glioblastoma, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors,hypothalamic glioma, breast cancer, male breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknownorigin, central nervous system lymphoma, cerebellar astrocytoma,malignant glioma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, acute lymphocyticand myelogenous leukemia, chronic myeloproliferative disorders,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,ependymoma, esophageal cancer. Ewing family tumors, extracranial germcell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastriccancer, gastrointestinal stromal tumor, extracranial germ cell tumor,extragonadal germ cell tumor, ovarian germ cell tumor, gestationaltrophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer,hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin lymphoma,hypopharyngeal cancer, hypothalamic and visual pathway glioma,intraocular melanoma, islet cell tumors. Kaposi sarcoma, kidney cancer,renal cell cancer, laryngeal cancer, lip and oral cavity cancer, smallcell lung cancer, non-small cell lung cancer, primary central nervoussystem lymphoma, Waldenstrom macroglobulinemia, malignant fibroushistiocytoma, medulloblastoma, melanoma. Merkel cell carcinoma,malignant mesothelioma, squamous neck cancer, multiple endocrineneoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplasticsyndromes, myeloproliferative disorders, chronic myeloproliferativedisorders, nasal cavity and paranasal sinus cancer, nasopharyngealcancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreaticcancer, parathyroid cancer, penile cancer, pharyngeal cancer,pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary cancer, plasma cell neoplasms,pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma. Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, choriocarcinoma, hematological neoplasm,adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumorsand germ cell tumors, or Wilms tumor. In some embodiments, the cancer islung cancer, breast cancer, prostate cancer, colorectal cancer, gastriccancer, liver cancer, pancreatic cancer, brain and central nervoussystem cancer, skin cancer, ovarian cancer, leukemia, endometrialcancer, bone, cartilage and soft tissue sarcoma, lymphoma,neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ celltumor.

As used herein, the term “treating” refers to the application oradministration of the delivery vehicle. the ceramide-therapeutic agentcomplex, or the composition comprising such to a subject in needthereof. “A subject in need thereof”, refers to an individual who has adisease, a symptom of the disease, or a predisposition toward thedisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the disease, the symptom of thedisease, or the predisposition toward the disease.

A “subject” to which administration is contemplated refers to a human(i.e., male or female of any age group, e.g., pediatric subject (e.g.,infant, child, or adolescent) or adult subject (e.g., young adult,middle-aged adult, or senior adult)) or non-human animal. In someembodiments, the non-human animal is a mammal (e.g., rodent (e.g., mouseor rat), primate (e.g., cynomolgus monkey or rhesus monkey),commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat,cat, or dog), or bird (e.g., commercially relevant bird, such aschicken, duck, goose, or turkey)). The non-human animal may be a male orfemale at any stage of development. The non-human animal may be atransgenic animal or genetically engineered animal.

In some embodiments, the subject is a companion animal (a pet). “Acompanion animal,” as used herein, refers to pets and other domesticanimals. Non-limiting examples of companion animals include dogs andcats; livestock such as horses, cattle, pigs, sheep, goats, andchickens; and other animals such as mice, rats, guinea pigs, andhamsters. In some embodiments, the subject is a research animal.Non-limiting examples of research animals include: rodents (e.g., rats,mice, guinea pigs, and hamsters), rabbits, or non-human primates.

In some embodiments, a “subject in need thereof” refers to a subjectthat needs treatment of a disease described herein.

Alleviating a disease includes delaying the development or progressionof the disease, or reducing disease severity. Alleviating the diseasedoes not necessarily require curative results. As used therein.“delaying” the development of a disease means to defer, hinder, slow,retard, stabilize, and/or postpone progression of the disease. Thisdelay can be of varying lengths of time, depending on the history of thedisease and/or individuals being treated. A method that “delays” oralleviates the development of a disease, or delays the onset of thedisease, is a method that reduces probability of developing one or moresymptoms of the disease in a given time frame and/or reduces extent ofthe symptoms in a given time frame, when compared to not using themethod. Such comparisons are typically based on clinical studies, usinga number of subjects sufficient to give a statistically significantresult.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a disease includes initial onset and/orrecurrence.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the isolated polypeptide orpharmaceutical composition to the subject, depending upon the type ofdisease to be treated or the site of the disease. This composition canalso be administered via other conventional routes, e.g., administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrastemal, intrathecal, intralesional, and intracranial injection orinfusion techniques. In addition, it can be administered to the subjectvia injectable depot mutes of administration such as using 1-, 3-, or6-month depot injectable or biodegradable materials and methods.

Some of the embodiments, advantages, features, and uses of thetechnology disclosed herein will be more fully understood from theExamples below. The Examples are intended to illustrate some of thebenefits of the present disclosure and to describe particularembodiments, but are not intended to exemplify the full scope of thedisclosure and, accordingly, do not limit the scope of the disclosure.

EXAMPLES Example 1. Test if the Oligosaccharide Domain of GM) isEssential for Intracellular Trafficking and Cargo Transport

Mucosal surfaces represent vast areas where host tissues are separatedfrom the environment only by a delicate but highly effective singlelayer of columnar epithelial cells, joined by tight junctions that areimpermeable to proteins and even small peptides. Proteinsnon-specifically taken up directly into the epithelial cell byendocytosis are generally transported to lysosomes for degradation. Sofar, the lack of rational and efficient methods to circumvent thisbarrier has prevented the application of most therapeutic proteins andpeptides for mucosal drug delivery.

Endothelial cells also form vast and highly restrictive single cellthick barriers that separate most tissues from the blood stream. Exceptfor the vasculature in the intestine, glomerulus, and liver, the healthynon-inflamed endothelial barrier strongly limits the permeability oflarge molecules; thus preventing access of many protein-based biologicsto cells of many tissues—even when the therapeutic proteins are appliedintravenously.

The pathway for large solutes (e.g., protein and peptide biologics) tocross simple epithelial and endothelial barriers with highly resistantintercellular tight junctions is by transcytosis—a process oftranscellular membrane trafficking that connects one surface ofpolarized cells with the other (FIG. 1 ). It has been described that theGM1 glycosphingolipid crosses epithelial barriers by transcytosis andthe structure of its ceramide domain dictates transport through thispathway. GM1 species containing non-native “short chain” fatty acids inthe ceramide domain was shown to allow for more efficient transportacross simple epithelial barriers, and some can release from the cellmembrane back into solution after transcytosis. It was also found thatthe “short-chain” GM1 glycosphingolipids can carry therapeutic peptides(GLP-1) across the intestinal epithelial barrier to deliver themsystemically. These in vivo studies show absorption far surpassing thebest currently achieved for oral delivery of biologics.

It was found in non-polarized cells that GM1-ceramides containing“kinked” cis-unsaturated C18:1 or C16:1 fatty acids, or non-native“short chain” C12:0 to C4:0 fatty acids, enter the sorting/recyclingendosome of epithelial cells for transport to various intracellulardestinations: including the recycling pathway and retrograde pathway toGolgi and ER. These lipids do not traffic into the lateendosome-lysosome pathway. In contrast, the GM1-sphingolipids with longsaturated fatty acid chains (C16:0 or longer) sort efficiently into lateendosomes and lysosomes (FIG. 19 ). The results are consistent withmodels for lipid sorting by molecular shape and membrane domains.

In polarized epithelial cells, another sorting event emerges from thesorting endosome and leads into the transcytotic pathway. This pathwayallows for the short- or unsaturated GM1-ceramides to cross the cell tothe opposite cell surface, thus breeching the epithelial barrier bytranscytosis (FIG. 1 ). It has been found that GM1 glycolipids can carrythe 80 kDa protein cholera toxin fully across polarized monolayers ofintestinal epithelial cells in culture, implicating a function for thispathway in protein absorption across the intestine in vivo.

Based on these results, the idea that the glycosphingolipids may beharnessed as trafficking vehicles for biologic drug delivery was tested.A all D-isomer amino acid reporter-peptide carrying a reactive aminooxyC-terminal residue for coupling to sialic acid of the glycoceramide GM1,a biotin group for rapid and high affinity isolation, and an alkynegroup for coupling to cargo (e.g., for detection via Alexa Fluor 488)was developed (FIG. 2 ).

Structure-function studies showed that certain novel short-chainceramide species allowed for transcellular transport (transcytosis) ofthe peptide-GM1 fusion molecule across epithelial monolayers in vitro asassessed biochemically (FIG. 3 ) and morphologically (FIG. 21 ). Studiesusing chemical inhibitors and genetic depletion of genes required fortranscytosis showed that the peptide-GM1 fusion molecules cross theepithelial monolayers by passing through the cell by transcytosis, andnot around the cell by paracellular “leak” (FIG. 22 ).

When applied orally (by gastric gavage) to mice in vivo, the peptide-GM1fusion molecules were absorbed across the intestine into the circulationas assessed in blood and in liver tissue (FIGS. 4A-4D). Uncoupledpeptides, however, are not absorbed. The absorption of the peptide-GM1fusion molecules in the nasal epithelium was observed, as assessedbiochemically in blood, and morphologically by imaging (FIG. 23 ).

Translational studies were carried out using the incretin hormone GLP-1as cargo. Human GLP1 is a cleavage product of proglucagon produced inpancreatic or intestinal enteroendocrine cells. Native GLP-1 possessesseveral physiological properties on glucose metabolism that make it aneffective treatment for diabetes mellitus type II. There also arereceptors for GLP-1 in the brain, and evidence that the molecule mayoperate in appetite control, suggesting other clinical applications. Inthis case, transport across the epithelial barrier is almost 100-foldabove the GLP-1 peptide alone. Fusion of the peptide hormone to thelipid carrier causes a very modest loss of hormone function as assessedby dose response in bioassay shown in panel to right (fusion molecule inRED is about a half-log less potent but still functional in picomolarrange). It was found that GLP-1 incretin function is retained afterfusion to the oligosaccharide domain of GM1 (FIG. 5A) (half-log loss inactivity). When tested in vitro, GLP-1 was transported across epithelialbarriers by the short-chain GM1-species nearly 100-fold above thatobserved for the peptide alone (FIG. 5B).

Consistent with the in vivo studies using the reporter-peptide-GM1fusion molecules, it was found that when applied to mice via gastricgavage, GLP-1-GM1 fusion molecules were absorbed and had effects overglucose metabolism, evidenced by reduced time to normalize blood sugarin glucose tolerance tests (FIG. 6 ). An assay was recently developed toconfirm biochemically that the GLP-1-GM1 fusion molecules are absorbedinto the blood (not shown). The GLP-1 peptide alone is not absorbed andhas no detectable effect on glucose metabolism.

Another structural feature of GM1 that might play a role in traffickingis the extracellular oligosaccharide head group. Further describedherein are studies designed to test if the oligosaccharide domain of GM1is essential for intracellular trafficking and cargo transport.Simplifying the lipid carrier may be important to clinical translation.It is tested herein if a simple glycoceramide easily harvested frombuttermilk can be used in place of GM1, and if the oligosaccharidedomain of GM1 can be eliminated completely, thus defining a simplifiedcore delivery molecule that can be fully and pragmatically synthesized.

Simplifying the glycoceramide structure would have two majorsignificances. First, this would allow for total chemical synthesis ofthe molecules and permit easier translation to the clinic. Second,structural elements of the oligosaccharide head group that affecttrafficking may be identified, enhancing the understanding of thestructure-activity relationship for the glycosphingolipids, and aidingin design of new peptide linkers that recapture the lostfunctionalities.

Some functionality of the sugar head groups are anticipated. At minimum,the oligosaccharide domain of GM1 is expected to function by trappingthe ceramide in the outer membrane leaflet, preventing flip-flop andrendering the molecule dependent on membrane trafficking fordistribution within and across cells, or by shaping the molecule. Suchfunctions, however, might be reproduced simply by attachment of peptideor protein cargo to the ceramide domain. Still, the oligosaccharide headgroup of GM1 might also participate in sorting reactions that drive GM1trafficking. If so, it may be possible to design peptide linkers thatrecover such trafficking functions. The testing of the oligosaccharidedomain and accommodation to its loss are the topics of this aim.

Initially, two simplified structural analogs of GM1 are tested:glucosylceramide (Glc-Cer-one sugar residue), and ceramide alone (nosugars). These lipids are commercially available with different ceramidestructures (Avanti Polar Lipids). Because ceramide (Cer) and Glc-Cerlack a sialic acid, this necessitates an alternative chemical strategyto link peptides onto the ceramide headgroup. All lipids are coupled tothe reporter peptide using copper-catalyzed “click” chemistry. BecauseCer and Glc-Cer lack a sialic acid, this necessitates an alternativechemical strategy to link peptides onto the ceramide headgroup. Detailsof the chemical reactions are shown in FIG. 7 . The primary hydroxyllocated on the ceramide (or on glucose ring of Glc-Cer) are oxidized toreact with peptides containing aminooxy reactive groups as inestablished method (12, 13). All compounds are confirmed for mass byMALDI mass spectrometry (or LC-MS) and NMR.

The transcytosis of Glc-Cer and Cer are tested in vitro using polarizedcell lines T84, Caco2 and MDCK. Testing the new peptide-ceramide fusionmolecules for transcytosis using the same methods described in FIG. 5 .Due to a possibility of being less soluble, the lipids are complexed toBSA (1:1) as described in Pagano et al. (43).

If reporter peptide fusions to ceramide alone mimic the GM1-traffickingplatform, it can be concluded that the sugar groups are dispensable,except perhaps for blocking the sphingolipid from membrane flip-flop tothe inner leaflet, a function presumably accommodated by thepeptide-linker and cargo. If the oligosaccharide domain contributesdecisively to trafficking function (and thus are required), peptidelinkers that would replace the functionality of the oligosaccharidedomains in trafficking can be designed. Due to the inherent difficultyin synthesizing complex O-linked oligosaccharides onto lipids,pseudo-glycopeptide linkers are synthesized using backbone amino acidscoupled to glucose, galactose or galactose-N-acetyl residues via serineside chains (44). FMoc-protected building block amino acids containingmonosaccharides are commercially available and will be used for solidphase peptide synthesis of short linear peptides containing up to 4-5sugar residues each (FIG. 8 ). Addition of the sugar groups mayreproduce the overall “bulk” and strong hydrophilicity imparted by theendogenous GM1 oligosaccharide, or function to display glucose orgalactose as ligands for extracellular lectins that could participate inthe lipid sorting events—such as that proposed for galectin 3 innon-clathrin mediated endocytosis (41). When necessary, additionalpeptides are synthesized in a branched manner (as observed with thenatural sialic acid residue), or cyclized to alter headgroup geometry toapproximate the GM1 oligosaccharide. The transcytosis assay can beperformed in a medium throughput manner, thus assess many differentpeptides constructs together.

If glycoceramide (single sugar) mimics results with the GM1-lipids, butceramide alone does not, it can be concluded that the sugar groupscontribute to glycosphingolipid sorting primarily by anchoring themolecules in the outer-leaflet of the membrane. In both cases, it ispossible that simplified sphingolipids can substitute for GM1 (or withredesigned peptide-sugar linkers).

The ability of ceramide alone or Glc-Cer to function as the GM1-lipidplatform allow more rapid translation to clinical application, as thesemolecules can be synthesized or readily purified from milk fats.

Example 2. Test if the Peptide Linker Between GM1 and Cargo can beDesigned to Release the Cargo after or During Transcytosis

Described herein is the design of a delivery vehicle that releases thecargo after transport. For instance, fusion of GM1 to the smallerpeptide hormone GLP-1 causes a 3 to 8-fold loss of function. While stillhighly potent (pM activity) and physiologically relevant, the GLP-1-GM1fusion molecule is still less potent than the native peptide. Here, twoapproaches to design the peptide linker so that it can release cargofrom GM1 after uptake into the cell or after arrival in the basolateralendosome were tested. One approach involved the incorporation of anester linkage in the peptide linker (as target for esterases increasedat sites of inflammation. e.g., leukocyte esterase, or carboxylesterasehCE-2 that is specific to gastrointestinal endosomes) (1, 45); and theother involved incorporation of a cleavage motif for the endosomalprotease furin (2-4).

Different furin cleavage motifs were tested for activity within thecontext of short peptide linker. The assay was designed ashigh-throughput using fluorescence energy transfer (FRET) by appendingpaired fluorophores at each end of the peptide to be tested. FIG. 9shows identification of an optimal sequence (FRET 7) for furthertesting. This motif was incorporated into the linker system describedherein and the peptide is synthesized, purified, and validated by massspec in large quantities. It is ready for coupling to cargo and GM1 fortesting in vitro and in vivo.

The FRET 7 furin cleavage motif or a cleavable ester linkage areincorporated into the reporter-peptide linker, and also with GLP-1incorporated as cargo. The GM1-C12:0 species are used as a fusionpartner because this lipid does not readily release from membranes andwill provide a more sensitive approach to test this technology. Theefficiency for cleavage are first tested in vitro using recombinantesterases or recombinant furin and analyzed by HPLC. If cleavage isconfirmed, the fusion molecules are tested for efficiency of cargotransport across highly resistant T84 epithelial barriers (as describedin FIG. 5 ). Control cargos are fused to GM1-C12:0 using non-cleavablelinkers. The fusion molecules are applied to apical surfaces of T84cells in the presence of serine protease inhibitors, BSA asserine-protease decoys, and ester-linked peptide decoys. Rates oftransepithelial transport will be compared using non-cleavable peptidelinkers as controls. An increase in transepithelial transport (orcytosolic delivery of cargo) above that achieved by the non-cleavablepeptide linkers suggests that cleavable linker technologies arefeasible.

REFERENCES

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Example 3. Fatty Acid Length & Double Bond Positioning Dictate EndosomalSorting of Glycosphingolipids

The ceramide structure plays a decisive role in determining the cellularfate of glycosphingolipids after endocytosis—explained by theirpropensity to sort into highly curved tubules and buds emerging from thesorting endosome. It was tested herein whether glycosphingolipid sortingdepends on molecular shape or on assembly into nano-domains (or both).GM1 species with saturated long chain fatty acids might a higherpropensity to self-assemble with cholesterol into nano-domains. Thesedomains might be recognized by cellular factors and sorted into thedegradative pathway; preventing entering the recycling endosomaltubules.

To investigate the role of double bond positioning & hydrocarbon chainlength of the ceramide fatty acid in lipid packing and subsequentdifferential endosomal sorting, GM1 isoforms with identicaloligosaccharide head groups but ceramides of different endogenousstructure were synthesized, by systematically increasing length anddouble bond position (C12:0 to C26:1).

The GM1 species with different ceramide structures were functionalizedwith a D-amino acid reporter peptide, containing a biotin andfluorophore group. Alternatively, a fluorophore was directly onto thesialic acid of the sugar head group. Lipids were purified by HPLC andstructures validated by LC/MS (FIG. 17 ).

Surface GM1 at steady state depends on lipid-sorting into highly curvedtubules of the recycling pathway and this can be detected usingfluorescent avidin probes. GM1 with saturated longer chain (>C14:0)fatty acid or long chain unsaturated fatty acid (>C24:1) are depletedfrom the plasma membrane, presumably sorted to the lysosome, suggestinginefficient entry into sorting tubules. GM1 containing a ceramide withC14:0 or C24:1 fatty acid have intermediate phenotype (FIG. 18 ).

Fluorescently-labeled GM1 in sorting tubules emerging from earlyendosomes (labeled with dextran and TFnR) was measured directly in A431cells and quantified. GM1 with short (<C14:0) or unsaturated (<C24:1)fatty acids escaped the degradative pathway and enter recyclingendosomal tubules. Depletion of cellular cholesterol by MbCD releasesGM1 species with long (>C14:0, ≥C24:1) fatty acids into recyclingendosomal tubules (FIG. 19 ).

Two additional endocytic pathways depend upon sorting into highly curvedtubules—transcytosis and retrograde, providing other quantitativemeasures for GM1 entry into sorting tubules. Transcytosis of individualGM1 species were measured in polarized MDCK cells detecting apical tobasolateral transport of GM1 with a fluorescent avidin probe. Retrogradetrafficking of individual GM1 species was measured using cAMP afterintoxification by cholera toxin as read out in GM1 negative cell lines.The toxin must traffic retrograde to the ER to induce cAMP. GM1 withlonger chain fatty acids (≥C14:0) or ceramides containing amono-unsaturated fatty acid (≥C22:1) inefficiently enter sorting tubulesand (presumably) are retained in the lysosomal pathway (FIG. 20 ). Theposition of the unsaturated bond suggests a decisive factor may beinteraction with cholesterol.

Methods of Examples 1-3 Synthesis of Peotide-GM1 Conjugates

A short reporter peptide (all-D isomer) was synthesized and chemicallylinked onto the headgroup of ceramide lipid containing a C2:0 fatty acidtail. The chemistry to do this was very difficult due to the nature ofthe components and insolubility on standard solvents. Many differentapproaches had been tried a strategy that worked well was found. The3-step synthetic approach is described herein.

Step 1: Oxidation of C2-Ceramide

A method called “Parikh-Doering” oxidation was used to oxidize theheadgroup of ceramide to a reactive aldehyde in organic solvent.Reaction mixture contains: (1) SO3Py: 190-400 mg/mL in dry DMSO; (2) C8glucosylceramide: 0.2-0.6M solution in dry DMSO with Triethylamine; and(3) Triethylamine: 7-17 equivalents. The resulting product was purifiedby solvent extraction and dried in a speed vac (FIG. 11 ).

Step 2: Reaction of Peptide Containing Aminooxy to the Aldehyde Group

The “LC9” (D-isomer) linker peptide was coupled using oxime-mediatedreductive amination (FIG. 12 ) in 80% DMF in 20% PBS pH6.9 in 500 μL.The reaction mixture contained 471 nmoles peptide, 1.642 nmoles oxidizedceramide, and 1 μL aniline. After overnight incubation, 5 mM sodiumcyanoborohydride was added to reduce the oxime bond formed and to makeit a permanently stable bond.

The resulting crude peptide-ceramide conjugates or purified conjugates(60% yield) were analyzed by HPLC (FIGS. 13A-13C). The products werealso analyzed by mass spectrometry to confirm the presence of thepeptide-ceramide conjugates (FIG. 13D).

Step 3: Copper Click Reaction of Alexa Fluor 488 to Ceramide-PeptideConjugate

Alexa Fluorophore containing an azide reactive group was reacted to thepeptide-ceramide conjugate via the N-terminal alkyne using Huisgencopper-catalyzed cycloaddition chemistry (FIG. 14 ). Briefly,Ceramide-peptide conjugate at 186 nmoles were reacted to 279 nmolesfluorophore in 50 mM Tris-C1 pH8, containing 100 mM ascorbate, 5 mMcopper sulfate. 0.06 mM TBTA and 1 mM TCEP. The products were analyzedand purified by semi-preparative HPLC (FIG. 15 ). Final molecular weightof the produce was 2,425.1 Da

Transcytosis in MDCK Cells

The in vitro transport (transcytosis) of the peptide-GM1 conjugatesacross a monolayer of MDCK cells, and in vivo transport across anintestinal or nasal barrier into the blood are assessed. The peptidealone are used as negative controls, and the GM1 analogs as positivecontrols. C6, and C12 fatty acid analogs of ceramide were also includedin the experiments as direct comparisons to the GM1 and GM3 species.

MDCK cells were seeded in 0.33 cm² inserts 3 days prior in mediacontaining HBSS and 100 mM defatted BSA. Electrical resistance waschecked on day of experiment.

Test Samples

-   -   1) peptide Control    -   2) GM1-C6:0-peptide    -   3) Peptide-C12 fatty acid    -   4) Ceramide-C2-

Media Preparation

-   -   Make: 27 mL T84-SF PLUS 1% dfBSA (add 270 mg dfBSA to 27 mL)    -   Make: 500 uL×50 samples=25 mL T84-SF PLUS 0.1% dfBSA (add 25 mg        to 25 mL)

Transwell Assay

-   -   1) Check electrical resistance by EVOM.    -   2) Prepare stock solutions above.    -   3) Wash cells apical and basolateral with HBSS, without FBS.        (dunk method)    -   4) EVOM the cells.    -   5) Replace apical with HBSS+0.1 uM dfBSA, basolateral with        HBSS+1% dfBSA.    -   6) Incubate again for 20 minutes. Re-check EVOM.    -   7) Wait another 20 minutes and remove/replace apical with 200□L        compound.    -   8) Continue incubation for 3 hours total.    -   9) Remove apical media to tubes.    -   10) Replace with apical media and recheck EVOM final.    -   11) Remove basolateral media to eppendorf tubes on ice.

Pull-down Assay

-   -   1) Washed Stretavidin Magnetic beads with TBS-T 4×, to remove        azide.    -   2) Resuspend heads in 800 uL TBS then aliquoted 50 uL out to        each tube    -   3) Add 1000 μL to streptavidin heads overnight at 4° C. with        rotation O/N covered in foil.    -   4) Remove media and wash 3×TBS-T.    -   5) Elute by addition of 220 ul 95% Formamide. 10 mM EDTA 0.4        mg/ml biotin—2 min@65° C.    -   6) Pipetted 100 uL of each sample ×2 on 96 well plate    -   7) Fluorescence was read out on a microplate reader for        Fluorescein channel, and against a standard curve.

Example 4. Pharmacokinetics and Bioavailability of Peptide-GM1-C6:0Fusion

The bioavailability of the peptide-GM1-C6:0 fusion molecule when appliednasally to mice and the pharmacokinetics are shown in FIG. 26 (means oftwo independent experiments). The dose was 2.5 nmol/kg for 7 week oldC57B/6J with an intranasal administration volume of 10 μL and anintraperitoneal injection volume of 200 μL. Nasal bioavailability ofC6-GM1 is 24.4%, and nasal bioavailability of the peptide alone is 1.3%.The 24% absorption compared to intraperitoneal injection of the samemolecule is high for mucosal absorption of therapeutic peptides.

Next, the bioavailability and pharmacokinetics of a peptide fused toGM-C4:0 was tested in vivo in rat. The fusion molecule was validated inmice before the experiment (FIG. 32A). The peptide alone or thepeptide-GM1-C4:0 conjugated was administered to rat via gastric route orvia intravenous injection. The result showed that there was noadsorption of gastric peptide-GM1-C4:0 conjugate. However, serumhalf-life of the peptide was strongly prolonged by fusing the peptide toGM1-C4:0, which is consistent with protection against degradation orexcretion by GM1-dependent trafficking into recycling endosomes ofendothelial and other cell types (liver, lymphocytes, spleen, etc.). Theresults showed that, on day one after intravenous injection, the levelof peptide-GM1-C4 fusion in rat serum was 5.6 nmol/kg and the level ofpeptide alone in rat serum was 10.8 nmol/kg (FIG. 32B). On day two afterintravenous injection, the level of peptide-GM1-C4 fusion in rat serumwas 14.4 nmol/kg and the level of peptide alone in rat serum was 12.8.8nmol/kg (FIG. 32C).

Example 5. Ceramides and Analogs as Delivery Vehicles

A panel of ceramides and ceramide analogs were synthesized (FIG. 27 ).Transcytosis of the ceramides and ceramide analogs in MDCKII cells wasevaluated, and the apparent permeability coefficient (Papp) values areshown in FIG. 28 . Transcytosis of the ceramides and ceramide analogs inT84 intestinal cells was also evaluated, and the apparent permeabilitycoefficient (Papp) values are shown in FIG. 29 . The result shows thatglycoceramide and ceramide alone are functional as delivery platformsand exhibited even more efficient than GM1.

Cer-C6 was tested along with a 4° C. temperature block to demonstrateevidence for the mechanism of transport by transcytosis. The ceramideanalog Diene C6 along (structure shown in FIG. 27 ) was also tested inthe same experiment. The results show that the low temperature reducedthe uptake of Cer-C6 and the Diene C6 ceramide analog by MDCK H cells,indicating that the transport was via transcytosis with a 4° C.temperature block (FIG. 30 ). This experiment was repeated withceramides of different fatty acid chain length (C4, C6. C8 (C8 resultsnot shown)), and with or without a sugar moiety. The results show thatceramide Cer-C6:0 carrier (without sugar) and the ceramide analog dienecarrier (C6:0) is effective in transporting across the MDCKII cells viatranscytosis, with the Cer-C6:0 being more effective. The glucoceramideC8:0 carriers also effectively transported via transcytosis (data nowshown).

Next, a ceramide with a C2 fatty acid chain and without a sugar moiety(Cer-C2) was tested. Peptide fused to Cer-C2 was able to transportacross monolayers of MDCKI cells in a transcytosis assay (FIG. 31 ). TheGM1-C6:0 molecule was used as a positive control, and a peptide fused toa fatty acid dodecyl-C12 was used as a negative control. Dodecyl-C12 didnot enable transport across MDCKII cells by transcytosis (FIG. 31 ).

A repeat experiment of transcytosis in MDCK cells is shown in FIG. 33 .Papp at 37° C. was paired with 4° C. to test for transport bytranscytosis (and not paracellular leak). Another independent experimentthat shows the ceramide alone Cer-C6:0 carrier is effective. Theceramide-like diene carrier (C6:0) also works, but is less effectivecompared to the ceramide-C6:0 species. The glucoceramide C8:0 carriersalso work and are probably as effective as ceramide C6:0 alone. Both aremore effective than the original fusion molecules using GM1 C6:0.

Further, whether a ceramide can be absorbed via the nose is tested. Areporter peptide was conjugated to a ceramide-C6 (Cer-C6) wasadministered to C57BL/6J mice (n=1, from Jackson Labs) at a dose of 2nmol/kg. GM1-C6 conjugated to the same reported peptide was used ascontrol. A total of 5 μl per nostril was administered over 5 minutesunder isoflurane. Blood was collected via cardiac puncture 15 minutespost dose and the amount of the conjugate was evaluated by pulling downwith magnetic streptavidin bcads followed by elution and platefluorescence measurement. The data showed that ceramide-C6-reporterpeptide conjugate can be absorbed via nose in mice, and that the peptideconjugated to Cer-C6 is absorbed more efficiently than peptideconjugated to GM1-C6 (FIG. 38 ).

Example 6. Synthesis of Peptide-Ceramide Conjugates as a Platform forProtein Drug Delivery

Diene-analogs (due to dehydration) and dihydro-analogs of ceramide havebeen underlined for clarity in this disclosure. Oximes may be a mix ofcis and trans geometric isomers. Oximes have been arbitrarily drawn ascis-isomers in FIG. 35 and as trans-isomers (likely the predominantisomer) in FIG. 36 and FIG. 37 . Some oximes have been reduced to thecorresponding O-alkylhydroxylamine analogs. (Data not shown). Reactionswere run in Wheaton vials with triangular magnets unless otherwisestated. Chromatography on flash silica gel 60 (230-400 mesh) at 35° C.unless otherwise stated. Analytical thin layer chromatography (TLC)utilized silica gel 60 on glass plates that were visualized by UV 254,and by phosphoric acid charring on a hot plate at 240° C. HPLCpurifications on RP-C3 columns eluting with acetonitrile/watercontaining 0.1% formic acid. Mass Spectral data from an AgilentTechnologies 6120 quadripole LC-MS.

Linker Chemistry and “diene”-analog of ceramide (dehydration compound)(2S,3R,4E)-1-(O-Triphenylmethyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol[1-trityl-C6-ceramide 2 (R═—C₅H₁₁)]. To a vigorously stirred solution ofC6-ceramide 1 (R═—C₅H₁₁) (30.3 mg, 7.62×10⁻⁵ mol), ethyldiisopropylamine(18 μL, 13 mg, 1.0×10⁻⁴ mol), and DMAP (1.1 mg, 9.0×10⁻⁶ mol) in 500 μLof dry CH₂Cl₂, was added trityl chloride (23.4 mg, 8.4×10⁵ mol) in 200μL of CH₂Cl₂ at mom temperature over 5 min. The reaction mixture wasstirred for 3 days. The mixture was concentrated and chromatographed togive 1-trityl-C6-ceramide 2 (R═—C5H₁₁) (32.0 mg, 5.00×10⁵ mol, 66%) as aclear and colorless viscous liquid that was homogeneous by TLC(90:10:0.1 CHCl₃/EtOAc/TEA R_(f) 0.42): ¹H NMR (DMSO-d₆).

(2S,3R,4E)-3-O-Benzoyl-1-(O-triphenylmethyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol[1-trityl-3-benzoyl-C6-ceramide 3 (R═—C₅H₁₁)]. To a vigorously stirredsolution of 1-trityl-C6-ceramide 2 (R═—C₅H₁₁) (9.5 mg, 1.5×10⁵ mol),ethyldiisopropylamine (13.1 μL, 9.7 mg, 7.5×10⁵ mol, 500 mol %), andcatalytic DMAP in 500 μL of dry toluene, was added benzoyl chloride (2.5mg, 1.8×10⁵ mol). After 1 day, additional ethyldiisopropylamine (13.1μL) and benzoyl chloride (2.5 mg) were added. The reaction was filteredthrough a plug of 0.2 g silica gel eluting with CHCl₃/EtOAc/TEA(97:3:0.1 CHCl₃/EtOAc/TEA). The filtrate containing product waschromatographed (97:3:0.1 CHCl₃/EtOAc/TEA) to give1-trityl-3-benzoyl-C6-ceramide 3 (R═—C₅H₁₁) (10.0 mg, 1.34×10⁻⁵ mol,89%) as a clear and colorless viscous liquid that was homogeneous by TLC(97:3:0.1 CHCl₃/EtOAc/TEA R_(f) 0.45): ¹H NMR (DMSO-d₆).

(2S,3R,4E)-3-O-Benzoyl-2-(N-hexanoylamino)-4-octadecene-1,3-diol[3-benzoyl-C6-ceramide 4a (R═—C₅H₁₁)]. To a stirred solution of1-trityl-3-benzoyl-C6-ceramide 3 (R═—C₅₁Hu) (10. mg, 1.3×10⁵ mol) in 2mL of CH₂Cl₂MeOH (1:1) was added a solution of p-toluenesulfonic acidmonohydrate 13.8 mg, 2.0×10 mol) in 0.5 mL of MeOH. After 3 days. CH₂Cl₂was added and the solution washed twice with 1 mL portions of 8.5 mg/mLaqueous NaHCO₃. The organic phase was then washed with H₂O, dried overNa₂SO₄, and chromatographed (96:4 CH₂Cl₂MeOH) to give3-benzoyl-C6-ceramide 4a (R═—C₅H₁₁) (6.5 mg, 1.3×10⁻⁵ mol, 100%) as awhite solid that was nearly homogeneous by TLC (96:4 CH₂Cl₂/MeOH, R_(f)0.20): ¹H NMR (DMSO-d₆): mass spectrum C_(3I)H₅₂NO₄ ⁺: m/z calculated502.4, observed 502.5.

Linker

(2S,3R,4E)-3-O-Benzoyl-1-O—(2-(2-(2-(4-formylbenzamido)ethoxy)ethoxy)ethan-1-carbamoyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol[1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C₅H₁₁)]. To astirred solution of triphosgene (5.9 mg, 2.0×10⁵ mol, 20 eq) in 0.2 mLof anhydrous CH₂C2 was added dropwise over 2 min a solution of3-benzoyl-C6-ceramide 4a (R═—C₅H₁₁) (1.5 mg, 3.0×10⁵ mol, 1 eq) in 0.2mL of anhydrous CH₂C12 containing ethyldiisopropylamine (22 μL, 16 mg,3.0×10⁻⁵ mol, 43 eq). After 2 h, the reaction mixture was distilledusing a stream of nitrogen gas blowing over the reaction mixture and outthrough a solution of aqueous ammonium hydroxide/H₂O/i-PrOH (1:1:1). Theresidue was redissolved in 0.1 mL of anhydrous CH₂Cl₂ to give a lightyellow solution. To this solution of crude chloroformate 5a (R═—C₅H₁₁)was added rapidly dropwise a freshly prepared solution ofAld-PEG2-ammonium trifluoracetate (3.9 mg, 9.9×10⁻⁶ mol, 3.3 eq.Broadpharm, CAS 2055013-56-2) in 0.1 mL of dry CH₂C12 containingethyldiisopropylamine (12 μL, 8.9 mg, 6.9×10⁵ mol, 7 eq). After 1 h 15min, the solvent was removed to give a yellow semi-solid that waschromatographed (96:4:0.1 CHCl₃/EtOAc/TEA) to give a nearly quantitativeyield of 1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C₅H₁₁) asa faint yellow semisolid that was nearly homogeneous by TLC (96:4:0.1CHCl₃/EtOAc/TEA. R_(f) 0.18): ¹H NMR (DMSO-d₆).

Linker

The linker already has aldehyde function.

Coupling with LC9 to Give the Oxime

LC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a (R═—C₅H₁₁). To asolution of 1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a (R═—C₅H₁₁)(1.5 mg, 1.9×10⁻⁶ mol) and LC9 (2.7 mg, 1.9×10⁻⁶ mol) in 800 μL of DMFwas added 1 μL of aniline and the reaction stirred overnight. HPLCpurification of the reaction mixture gaveLC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a (R═—C₅H₁₁); massspectrum C₁₀₆H₁₆₃N₂₄O₄S⁺: m/z calculated for (M+2H)⁺² 1118.6, observed1118.9.

The “click” reaction with AF488-azide which is a copper(I)-catalyzedazide-alkyne cycloaddition (CuAAC Reaction) to give the correspondingpredominantly 1,4-substituted-1,2,3-triazole (as drawn in FIG. 37 ,though may contain some 1.5-substituted triazole).AF488-LC9-oxime-PEG2-carbamoyl-C6-ceramide-3 benzoate 9a (R═—C₅H₁₁),mass spectrum m/z calculated for (M+2H)⁺², observed.

Diene Oxidation of Primary Alcohol to Aldehyde

To a solution of C6-ceramide-3-benzoate 4a (R═—C₅H₁₁) (1 mg, 2×10⁻⁶ mol)in 0.2 mL of dry CH₂Cl₂, was added a 0.30 M solution of Dess-Martinperiodinane (13 μL, 4×10⁻⁶ mol) dropwise over 1 min during which timethe solution remained homogeneous. After 10 min, a vortex-mixed solutionof H₂O (0.04 μL, 0.04 mg, 2×10⁻⁶ mol) in 40 μL of CH₂Cl₂ was addeddropwise over 10 min during which time the reaction mixture becamecloudy. The reaction mixture was vigorously stirred for an additional1.5 h. The reaction mixture was then washed with 0.2 mL of 1:1 saturatedaqueous NaHCO₃/15% Na₂S₂O₃, dried over Na₂SO₄, filtered through a 0.45μm GHP filtration cartridge, and the solvent removed to give a lightyellow viscous oil that contained the corresponding aldehyde by TLC(90:10:0.1 CHCl₃/EtOAc/TEA. R_(f) 0.66) and was used immediately.

Diene Coupling with LC9 to Give the Oxime

LC9-oxime-C6-ceramide-diene 10 (R═—C₅H₁₁). To a solution of the crudealdehyde (0.5 mg, 1×10⁻⁶ mol) and LC9 (1.7 mg, 1.2×10⁻⁶ mol) in 1 mL ofDMF was added 1 μL of aniline and the reaction stirred overnight. HPLCpurification of the reaction mixture gave LC9-oxime-C6-ceramide-dieneanalog 10 (R═—C₅H₁₁) (0.4 mg, 2×10⁻⁷ mol): mass spectrumC₈₄H₁₃₇N₂₂O₂₀S⁺: m/z calculated for (M+2H)⁺² 903.5, observed 903.8,calculated for (M+3H)⁺³ 602.7, observed 603.1.

Diene

The “click” reaction with AF488-azide which is a copper(I)-catalyzedazide-alkyne cycloaddition (CuAAC Reaction) to give the correspondingpredominantly 1,4-substituted-1,2,3-triazole (as drawn in FIG. 37 ,though may contain some 1.5-substituted triazole).AF488-LC9-oxime-C6-ceramide-diene analog 13 (R═—C₅H₁₁). A solution ofLC9-oxime-C6-ceramide-diene analog 10 (R═—C₅H₁₁) (0.4 mg, 2×10⁻⁷ mol).Alexa Fluor® 488 (0.38 mg, 4.4×10⁻⁷ mol),tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (6×10 mol),tris(2-carboxyethyl)phosphine (1×10⁻⁶ mol), ascorbic acid (1×10⁴ mol).Cu(II)SO₄ (5×10⁻⁶ mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, wasstirred overnight protecting from light. The reaction mixture wascentrifuged, and the colored precipitate was purified by HPLC to giveAF488-LC9-oxime-C6-ceramide-diene analog 13 (R═—C₅H₁₁) (14 μg, 5.7×10⁻⁹mol, 2.8% yield); mass spectrum C₁₁₁H₁₆₃N28O₃₀S₃ ⁺: m/z calculated for(M+2H)⁺² 1232.6, observed 1232.5, calculated for (M+3H)+³ 822.1,observed 822.2.

AF488-LC9-Oxime-C6 Ceramide C6

(2S,3R,4E)-3-O-Triethylsilyl-2-(N-hexanoylamino)-4-octadecene-1,3-diol[3-TES-C6-ceramide 4b (R═—C₅H₁₁)]. To a stirred solution of C6-ceramide1 (R═—C₅H a) (30.3 mg, 7.62×10⁻⁵ mol), ethyldiisopropylamine (150 μL,111 mg, 8.5×10⁴ mol), and DMAP (9.5 mg, 7.8×10⁻⁵ mol) in 1.5 mL of dryCH₂Cl₂, was added triethylsilyl chloride (12 μL, 11 mg, 7.3×10⁻⁵ mol)dropwise at room temperature over 2 min. The reaction mixture wasstirred for 1.5 h. The mixture was concentrated and chromatographed(90:10:0.1 CHCl₃/EtOAc/TEA) first eluting 1,3-diTES-C6-ceramideR═—C₅H₁₁) followed by 1-TES-C6-ceramide (R═—C₅H₁₁), then the desired3-TES-C6-ceramide 4b (R═—C₅H₁₁) as the minor product (3.7 mg, 7.2×10⁻⁶mol, 9.4%) as a clear and colorless viscous liquid that was homogeneousby TLC (90:10:0.1 CHCl₃/EtOAc/TEA R_(f) 0.15): ¹H NMR (DMSO-d₆); massspectrum C₃₀H₆₂NO₃Si⁺: m/z calculated 512.5, observed 512.4.

C6

Step One is oxidation of primary alcohol to aldehyde. To a solution ofC6-ceramide-3-TES 4b (R═—C₅H₁₁) (2.2 mg, 4.3×10⁻⁶ mol) in 0.2 mL of dryCH₂C₂, was added a 0.30 M solution of Dess-Martin periodinane (22 μL,5.7×10⁻⁶ mol, 1.5 eq) dropwise over 1 min during which time the solutionremained homogeneous. After 10 min, a vortex-mixed solution of H₂O (0.13μL, 0.13 mg, 7.2×10⁻⁶ mol) in 130 μL of CH₂Cl₂ was added dropwise over10 min during which time the reaction mixture became cloudy. Thereaction mixture was vigorously stirred for an additional 15 min. Thereaction mixture was then washed with 0.2 mL of 1:1 saturated aqueousNaHCO₃/15% Na₂S203, dried over Na₂SO₄, filtered through a 0.45 μm GHPfiltration cartridge, and the solvent removed to give a light yellowviscous oil that contained the corresponding aldehyde by TLC (90:10:0.1CHCb3/EtOAc/TEA, R_(f) 0.62) and was used immediately.

C6

Step Two is the coupling with LC9 to give the oxime.LC9-oxime-C6-ceramide-3-TES 11a (R═—C₅H₁₁). To a solution of the crudealdehyde (2 mg, 4×10⁻⁶ mol) and LC9 (6.2 mg, 4.3×10⁻⁶ mol) in 1 mL ofDMF was added 1 μL of aniline and the reaction stirred overnight. HPLCpurification of the reaction mixture gave LC9-oxime-C6-ceramide-3-TES11a (R═—C₅H₁₁); mass spectrum C₉₀H₁₅₃N₂₂O₂₁SSi⁺: m/z calculated for(M+2H)⁺² 969.6, observed 970.5, calculated for (M+3H)*³ 646.7, observed647.2.

C6

Step Three is removal of the TES, LC9-oxime-C6-ceramide 11b (R═—C₅H₁₁).A solution of LC9-oxime-C6-ceramide-3-TES 11a (R═—C₅H₁₁) in 80:20 aceticacid/H₂O was stirred for two hours. Solvents were removed bylyophilization. HPLC purification of the reaction mixture gaveLC9-oxime-C6-ceramide 11b (R═—C₅H₁₁) (1.0 mg, 5.5×10⁻⁷ mol); massspectrum C₈₄H₁₃₉N₂₂O₂₁S⁺: m/z calculated for (M+2H)⁺² 912.5, observed912.7, calculated for (M+3H)⁺³ 608.7, observed 609.1.

C6

Step Four is the “click” reaction with AF488-azide which is acopper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to givethe corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawnin FIG. 37 , though may contain some 1,5-substituted triazole).

AF488-LC9-oximc-C6-ceramide 14b (R═—CH₅H₁₁). A solution ofLC9-oximc-C₆-ceramide 11b (R═—C₅H₁₁) (1.0 mg, 5.5×10⁻⁷ mol), AlexaFluor® 488 (0.71 mg, 8.2×10⁻⁷ mol),tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine 16×10⁻⁸ mol),tris(2-carboxyethyl)phosphine (1×10⁻⁶ mol), ascorbic acid (1×10⁻⁴ mol),Cu(II)SO₄ (5×10⁻⁶ mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, wasstirred overnight protected from light. The reaction mixture wascentrifuged, and the colored precipitate was purified by HPLC to giveAF488-LC9-oxime-C6-ceramide 14a (R═—C₅H₁₁) (100 □g, 6.2×10⁻⁸ mol), massspectrum Cu₁₁₁H₁₆₅N₂₈O₃₁S₃ ⁺: m/z calculated for (M+2H)⁺² 1241.1,observed 1241.6, calculated for (M+3H)+³ 827.7, observed 828.2.

AF488-LC9-Oxime-C18 Ceramide C18

(2S,3R,4E)-3-O-Triethylsilyl-2-(N-octadecanoylamino)-4-octadecene-1,3-diol[3-TES-C18-ceramide 4b (R═—C₁₇H₃₅)]. To a stirred solution ofC18-ceramide 1 (R═—C₁₇H₃₅) (41.2 mg, 7.28×10⁻⁵ mol),ethyldiisopropylamine (150 μL, 111 mg, 8.5×10⁻⁴ mol), and DMAP (12.7 mg,1.04×10 mol) in 1.5 mL of dry CH₂Cl₂, was added triethylsilyl chloride(14 μL, 13 mg, 8.6×10⁻⁵ mol) dropwise at room temperature over 2 min.The reaction mixture was stirred for 1.5 h. The mixture was concentratedand chromatographed (90:10:0.1 CHCl₃/EtOAc/TEA) first eluting1,3-diTES-C18-ceramide R═—C₁₇H₃₅) followed by 1-TES-C₁₈-ceramide(R=—C₁₇H₃₅), then the desired 3-TES-C18-ceramide 4b (R═—C₁₇H₃₅) as theminor product (2.5 mg, 3.7×10⁻⁶ mol, 5.1%) as an amorphous white solidthat was homogeneous by TLC (90:10:0.1 CHCl₃/EtOAc/TEA R_(f) 0.15): ¹HNMR (DMSO-d₆).

C18

Step One is oxidation of primary alcohol to aldehyde. To a solution of3-TES-C18-ceramide 4b (R═—C₁₇H₃₅) (2.5 mg, 3.7×10⁻⁶ mol) in 0.2 mL ofdry CH₂Cl₂, was added a 0.30 M solution of Dess-Martin periodinane (19μL, 5.7×10⁻⁶ mol, 1.5 eq) dropwise over 1 min during which time thesolution became cloudy. After 10 min, a vortex mixed solution of H₂O(0.11 μL, 0.11 mg, 6.1×10⁻⁶ mol) in 110 μL of CH₂Cl₂ was added dropwiseover 10 min. The reaction mixture was vigorously stirred for anadditional 15 min. The reaction mixture was then washed with 0.2 mL of1:1 saturated aqueous NaHCO₃/15% Na₂S₂O₃, dried over Na₂SO₄, filteredthrough a 0.45 μm GHP filtration cartridge, and the solvent removed togive yellow viscous oil that was highly homogeneous aldehyde by TLC(90:10:0.1 CHCl₃/EtOAc/TEA. R_(f) 0.59) and was used immediately.

C18

Step Two is the coupling with LC9 to give the oxine with protectinggroup removal. LC9-oxime-C18-ceramide 11e (R═—C₁₇H₃₅). To a solution ofthe crude aldehyde (2.5 mg, 3.7×10⁻⁶ mol) and LC9 (5.3 mg, 3.7×10⁻⁶ mol)in 1 mL of DMF was added 1 μL of aniline and the reaction stirredovernight. HPLC purification of the reaction mixture gave only a smallamount of LC9-oxime-C18-ceramide-3-TES 11d (R═—C₁₇H₃₅), but most of theTES had fallen off and gave LC9-oxime-C18-ceramide 11e (R═—C₁₇H₃₅) (3.9mg, 2.0×10⁻⁶ mol, 54% yield) as a white solid: ¹H NMR (DMSO-d₆): massspectrum C₉₆H₁₆₃N₂₂O₂₁S⁺: m/z calculated for (M+2H)⁺² 996.6, observed996.9, calculated for (M+3H)⁺³ 664.7, observed 665.1.

C18

Finally, the “click” reaction with AF488-azide which is acopper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to givethe corresponding predominantly 1,4-substituted-1,2,3-triazole (as drawnin FIG. 37 , though may contain some 1.5-substituted triazole).

AF488-LC9-oxime-C18-ceramide 14b (R═—C₁₇H₃₅). A solution ofLC9-oxime-C18-ceramide 11e (R═—C₁₇H₃₅) (0.64 mg, 5.5×10⁻⁴ mol). AlexaFluor® 488 (0.71 mg, 4.9×10⁷ mol),tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (6×10⁻⁸ mol),tris(2-carboxyethyl)phosphine (1×10⁻⁴ mol), ascorbic acid (1×10⁻⁴ mol),Cu(II)SO₄ (5×10⁻⁶ mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, wasstirred overnight protected from light. The reaction mixture wascentrifuged, and the colored precipitate purified by HPLC to giveAF488-LC9-oxime-C18-ceramide 14b (R═—C₁₇H₃₅); mass spectrumC₁₂₃H₁₈₉N₂₈O₃₁S₃ ⁺: m/z calculated for (M+2H)⁺² 1325.7, observed 1325.6,calculated for (M+3H)⁺³ 884.1, observed 884.8.

AF488-LC9-Oxime-Dihydroceramide Dihydro

(2S,3R)-3-O-Triethylsilyl-2-(N-hexanoylamino)-octadecane-1,3-diol[3-TES-C6-dihydroceramide 7b (R═—C₅H₁₁)]. To a stirred solution ofC6-dihydroceramide 7a (R═—C₅H₁₁) (27.8 mg, 6.96×10⁻⁵ mol),ethyldiisopropylamine (280 μL, 208 mg, 1.61×10⁻³ mol), and DMAP (12 mg,9.8×10⁻⁵ mol) in 1.5 mL of dry CH₂Cl₂, was added triethylsilyl chloride(14 μL, 13 mg, 8.6×10⁻⁵ mol) dropwise at room temperature over 2 min.The reaction mixture was stirred for 1.5 h. The mixture was concentratedand chromatographed (90:10:0.1 CHCl/EtOAc/TEA) first eluting1,3-diTES-C6-dihydroceramide (R═—C₅H₁₁) followed by1-TES-C6-dihydroceramide (R═—C₅H₁₁), then the desired3-TES-C6-dihydroceramide 7b (R═—C₅H₁₁) as the minor product 12.7 mg,5.2×10⁻⁶ mol, 7.5%) as a white waxy solid that was homogeneous by TLC(90:10:0.1 CHCl/EtOAc/TEA, R_(f) 0.14): ¹H NMR (DMSO-d₆).

Dihydro

Step One is oxidation of primary alcohol to aldehyde. To a solution ofC6-dihydroceramide-3-TES 7b (R═—C₅H₁₁) (2 mg, 4×10⁻⁶ mol) in 0.2 mL ofdry CH₂Cl₂, was added a 0.30 M solution of Dess-Martin periodinane (13μL, 3.9×10⁻⁶ mol, 1.5 eq) dropwise over 1 min during which time thesolution remained homogeneous. After 10 min, a vortex-mixed solution ofH₂O (0.079 μL, 0.079 mg, 4.4×10⁻⁶ mol) in 10 μL of CH₂Cl₂ was addeddropwise over 10 min during which time the reaction mixture becamecloudy. The reaction mixture was vigorously stirred for an additional 30min. The reaction mixture was then washed with 0.2 mL of 1:1 saturatedaqueous NaHCO₃/15% Na₂S₂O3, dried over Na₂SO₄, filtered through a 0.45μm GHP filtration cartridge, and the solvent removed to give a lightyellow viscous oil that contained the corresponding aldehyde by TLC(90:10:0.1 CHCl₃/EtOAc/TEA. R_(f) 0.71) and was used immediately.

Dihydro

Step Two is the coupling with LC9 to give the oxime with protectinggroup removal. LC9-oxime-C6-dihydroceramide 12b (R═—C₅H₁₁). To asolution of the crude aldehyde (2 mg, 4×10⁻⁶ mol) and LC9 (5.6 mg,3.9×10⁻⁶ mol) in 1 mL of DMF was added 1 μL of aniline and the reactionstirred overnight. HPLC purification of the reaction mixture gave only asmall amount of protecting group removed LC9-oxime-C6-dihydroceramide12b (R═—C₅H₁₁); mass spectrum C₈₄H₁₄₁N₂₂O₂₁S⁺: m/z calculated for(M+2H)⁺² 913.5, observed 913.7, calculated for (M+3H)⁺³ 609.4, observed609.7.

All publications, patents, patent applications, publication, anddatabase entries (e.g., sequence database entries) mentioned herein.e.g., in the Background, Summary, Detailed Description, Examples and/orReferences sections, are hereby incorporated by reference in theirentirety as if each individual publication, patent, patent application,publication, and database entry was specifically and individuallyincorporated herein by reference. In case of conflict, the presentapplication, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of theembodiments described herein. The scope of the present disclosure is notintended to be limited to the above description, but rather is as setforth in the appended claims.

Articles such as “a.” “an.” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between two or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present, unless indicated to the contrary or otherwiseevident from the context. The disclosure of a group that includes “or”between two or more group members provides embodiments in which exactlyone member of the group is present, embodiments in which more than onemembers of the group are present, and embodiments in which all of thegroup members are present. For purposes of brevity those embodimentshave not been individually spelled out herein, but it will be understoodthat each of these embodiments is provided herein and may bespecifically claimed or disclaimed.

It is to be understood that the disclosure encompasses all variations,combinations, and permutations in which one or more limitation, element,clause, or descriptive term, from one or more of the claims or from oneor more relevant portion of the description, is introduced into anotherclaim. For example, a claim that is dependent on another claim can bemodified to include one or more of the limitations found in any otherclaim that is dependent on the same base claim. Furthermore, where theclaims recite a composition, it is to be understood that methods ofmaking or using the composition according to any of the methods ofmaking or using disclosed herein or according to methods known in theart, if any, are included, unless otherwise indicated or unless it wouldbe evident to one of ordinary skill in the art that a contradiction orinconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that every possible subgroup of the elements is alsodisclosed, and that any element or subgroup of elements can be removedfrom the group. It is also noted that the term “comprising” is intendedto be open and permits the inclusion of additional elements or steps. Itshould be understood that, in general, where an embodiment, product, ormethod is referred to as comprising particular elements, features, orsteps, embodiments, products, or methods that consist, or consistessentially of, such elements, features, or steps, are provided as well.For purposes of brevity those embodiments have not been individuallyspelled out herein, but it will be understood that each of theseembodiments is provided herein and may be specifically claimed ordisclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in some embodiments, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.For purposes of brevity, the values in each range have not beenindividually spelled out herein, but it will be understood that each ofthese values is provided herein and may be specifically claimed ordisclaimed. It is also to be understood that unless otherwise indicatedor otherwise evident from the context and/or the understanding of one ofordinary skill in the art, values expressed as ranges can assume anysubrange within the given range, wherein the endpoints of the subrangeare expressed to the same degree of accuracy as the tenth of the unit ofthe lower limit of the range.

Where websites are provided. URI, addresses are provided asnon-browser-executable codes, with periods of the respective web addressin parentheses. The actual web addresses do not contain the parentheses.

In addition, it is to be understood that any particular embodiment ofthe present disclosure may be explicitly excluded from any one or moreof the claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the disclosure, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

What is claimed is:
 1. A delivery vehicle comprising a ceramide and anagent to be delivered, wherein the ceramide: (a) does not comprise afatty acid; or (b) comprises a fatty acid of C1-C28; and wherein theagent is attached to the ceramide: wherein the ceramide does notcomprise an oligosaccharide; and wherein the agent is not a nucleicacid.
 2. The delivery vehicle of claim 1, wherein the ceramide is aceramide analog. 3.-4. (canceled)
 5. The delivery vehicle of claim 1,wherein the ceramide is a glycoceramide, wherein the sugar of theglycoceramide is a simple sugar.
 6. The delivery vehicle of claim 5,wherein the simple sugar is glucose, galactose, fructose, or GalNac. 7.The delivery vehicle of claim 6, wherein the agent to be delivered isattached to the sugar.
 8. The delivery vehicle of claim 1, wherein nosugar is attached to the ceramide.
 9. (canceled)
 10. The deliveryvehicle of claim 8, wherein the agent to be delivered is attached to theprimary or secondary hydroxyl group of the ceramide.
 11. The deliveryvehicle of claim 1, wherein the agent to be delivered is attached to theceramide via a linker. 12.-21. (canceled)
 22. The delivery vehicle ofclaim 1, wherein the ceramide comprises a fatty acid of C1-C6. 23.-25.(canceled)
 26. The delivery vehicle of claim 1, wherein the ceramidecomprises a fatty acid of C7-C28. 27.-31. (canceled)
 32. The deliveryvehicle claim 1, wherein the agent to be delivered is selected from thegroup consisting of proteins, peptides, polysaccharides andcarbohydrates, lipids, glycoproteins, small molecules, synthetic organicand inorganic drugs exerting a biological effect when administered to asubject, and combinations thereof.
 33. The delivery vehicle of claim 1,wherein the agent to be delivered is a therapeutic agent.
 34. Thedelivery vehicle of claim 33, wherein the therapeutic agent is ananti-inflammatory agent, a vaccine antigen, a small molecule drug, ananti-cancer drug or chemotherapeutic drug, a clotting factor, a hormone,a steroid, a cytokine, an antibiotic, an antibody, a ScFv, a nanobody, avaccine adjuvant, an agent that induces immune tolerance, or a drug forthe treatment of a cardiovascular disease, an infectious disease, anautoimmune disease, allergy, a blood disorder, a metabolic disorder, askin disease, an eye disease, a lysosomal storage disease, or aneurological disease. 35.-36. (canceled)
 37. The delivery vehicle ofclaim 32, wherein the protein or peptide is an antibody, a ScFv, or ananobody. 38-93. (canceled)
 94. A method of delivering an agent into acell, across an epithelial barrier, or across an endothelial barrier,the method comprising contacting a delivery vehicle with the cell, theepithelial barrier, or the endothelial lumenal surface, under conditionsappropriate for uptake of the delivery vehicle or the agent into thecell or absorption of the delivery vehicle or the agent across themucosal surface or the endothelial surface, wherein the delivery vehiclecomprises a ceramide and the agent, wherein the ceramide: (a) does notcomprise a fatty acid: or (b) comprises a fatty acid of C1-C28; whereinthe agent is attached to the ceramide; and wherein the ceramide does notcomprise an oligosaccharide. 95.-97. (canceled)
 98. A method ofenhancing the half-life of an agent in a subject, the method comprisingadministering to the subject a delivery vehicle comprising a ceramideand the agent, wherein the ceramide: (a) does not comprise a fatty acid;or (b) comprises a fatty acid of C1-C28; wherein the agent is attachedto the ceramide; and wherein the ceramide does not comprise anoligosaccharide.
 99. A method of treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject an effective amount of a delivery vehicle comprising a ceramideand an agent to be delivered, wherein the ceramide: (a) does notcomprise a fatty acid; or (b) comprises a fatty acid of C1-C28; whereinthe agent is attached to the ceramide; and wherein the ceramide does notcomprise an oligosaccharide. 100.-103. (canceled)
 104. The method ofclaim 94, wherein the ceramide is a glycoceramide, and wherein the sugarof the glycoceramide is a simple sugar.
 105. The method of claim 104,wherein the agent to be delivered is attached to the sugar.
 106. Themethod of claim 94, wherein no sugar is attached to the ceramide.